Work described in this thesis was aimed to further understand the role of macrophages in acute cellular rejection of transplanted kidneys, and perform a pre-clinical assessment of the localization... Show moreWork described in this thesis was aimed to further understand the role of macrophages in acute cellular rejection of transplanted kidneys, and perform a pre-clinical assessment of the localization and efficacy of liposomal prednisolone. First, we explored the role of anti-inflammatory macrophages in the onset of acute cellular rejection using human biopsy samples. Here, we discovered that the presence of high levels of CD163+ macrophages was associated with a lower risk of rejection, in a population of patients with a high rate of delayed graft function. Subsequently, we identified the ability of liposomal prednisolone to target macrophages in the kidney using in vitro models of human macrophages, and their ability to accumulate in inflamed kidney tissue in vivo rat models of ischemia reperfusion injury in the kidney. Finally, we evaluated the treatment efficacy of liposomal prednisolone in a mouse model of cellular rejection following kidney transplantation. Here, we were able to show an improved efficacy of liposomal prednisolone in treating rejection, when compared to treatment with free prednisolone. Show less
This thesis demonstrates that kidneys from DCD donors can be fully embraced, and that the perception of DCD kidney transplantation being inferior to DBD kidney transplantation – mainly motivated by... Show moreThis thesis demonstrates that kidneys from DCD donors can be fully embraced, and that the perception of DCD kidney transplantation being inferior to DBD kidney transplantation – mainly motivated by concerns regarding high incidences of DGF and EGL – is no longer justified. One explanation for this phenomenon is that outcomes of transplanted DCD kidneys have improved over time, with a similar incidence of EGL following DBD and DCD kidney transplantation in the current era. Another explanation is that both donor types have different biological responses to DGF: whilst DGF severely impacts on DBD graft survival, DGF has no impact on DCD graft survival. This difference relates to donor type-specific regulation of resilience and pro-inflammatory pathways benefitting the DCD graft and its outcomes. As such, the persistent high incidence of DGF in DCD grafts should not be regarded an impediment toward the use of these donor kidneys.This thesis additionally explores a series of physiological and methodological contrasts between preclinical and clinical I/R injury, that all may contribute to the impaired translatability of preclinical studies in clinical practice. Awareness of these pitfalls may help to improve study designs in future research, bringing us one step closer towards bridging the translational gap. Show less
This thesis was aimed at optimizing immunosuppressive therapy in kidney transplant recipients using pharmacometric models. Kidney transplantation comprises the preferred treatment strategy for... Show moreThis thesis was aimed at optimizing immunosuppressive therapy in kidney transplant recipients using pharmacometric models. Kidney transplantation comprises the preferred treatment strategy for patients with end-stage kidney disease. Its clinical success is challenged by graft rejection, necessitating lifelong immunosuppressive therapy to accommodate host-graft adaptation. Herein, achievement of balanced immunosuppression is vital for optimal outcomes, but is complicated by pharmacokinetic variability of the immunosuppressants. Currently, therapeutic drug monitoring (TDM)-guided dose individualization is conducted in an effort to achieve immunosuppressant exposure with adequate rejection prophylaxis and minimal toxicity. However, TDM target attainment rates are low and graft rejection and toxicity are observed in patients with on-target immunosuppressant exposure, indicating a need for further improvement. Pharmacometrics harnesses options to modernize this endeavor, allowing for model-based prediction of individual pharmacokinetic behavior and dosage requirements from patient characteristics and pharmacokinetic observations. We reviewed the current state of pharmacometrics in kidney transplantation, developed pharmacometric models for alemtuzumab and iohexol, externally evaluated a model-based dosing tool for everolimus, and combined pharmacometrics with microsampling to enable remote monitoring of immunosuppressant exposure and kidney function, simultaneously. Our research underlines the broad applicability of pharmacometrics and provides an impulse for future research to further optimize immunosuppressive therapy in kidney transplantation. Show less
This dissertation aimed to broaden our understanding of patient-relevant outcomes after kidney transplantation with a special focus on the patients’ perspectives. By conducting different studies in... Show moreThis dissertation aimed to broaden our understanding of patient-relevant outcomes after kidney transplantation with a special focus on the patients’ perspectives. By conducting different studies in Dutch kidney transplant recipients and by synthesizing existing evidence, we explored several post-transplant patient-relevant outcomes (e.g. health-related quality of life, symptom burden, illness perceptions, medication non-adherence and hospital readmission) and investigated pathways to improve these outcomes, hereby adding to the existing body of knowledge in kidney transplantation. In Chapter 1, we briefly introduced the status quo of patient-relevant outcomes in kidney transplant recipients, discussed potential pathways to improve patient-relevant outcomes in kidney transplant recipients, and provided an overview of the studies presented in this dissertation. In Chapter 7, we provided a summary of our main findings from different studies described in Chapter 2 - 6, the clinical implication of our main findings, and suggestions for future research. Show less
In part 1 of the thesis Predicting Outcomes in Patients with Kidney Disease, key differences between etiological and prediction research are explored and it is shown that observational research... Show moreIn part 1 of the thesis Predicting Outcomes in Patients with Kidney Disease, key differences between etiological and prediction research are explored and it is shown that observational research often conflates etiology and prediction which leads to incorrect causal conclusions. A framework for the external validation of prognostic models is provided and it is shown how competing events can be dealt with when externally validating a time-to-event prognostic model. These results are applicable to many clinical research fields, including nephrology as exemplified in part 2. Within the six applied chapters in part 2, prediction models for various adverse outcomes in patients with advanced kidney disease are identified, validated and developed. The thesis provides a broad overview of prognostic model applications in patients with chronic kidney disease, including comprehensive external validation studies for kidney failure prediction models, mortality prediction models and graft failure prediction models. Models to predict mortality on conservative care and dialysis and models to predict adverse outcomes after kidney transplantation were developed and validated. These results may improve shared decision-making processes and individualized medicine for patients with kidney disease. Show less
In chapters 2, 3 and 4, novel biomarkers IGFPB7, TIMP-2 and long noncoding RNAs were studied in order to find new diagnostic possibilities for early recognition and diagnosis of injury in native... Show moreIn chapters 2, 3 and 4, novel biomarkers IGFPB7, TIMP-2 and long noncoding RNAs were studied in order to find new diagnostic possibilities for early recognition and diagnosis of injury in native and transplanted kidneys. In chapter 2, diabetic nephropathy was found to be associated with higher levels of circulating TIMP-2, which did not normalize after simultaneous pancreas-kidney transplantation. In chapter 3, we found that four circulating long noncoding RNAs associated with diabetic nephropathy and did normalize after simultaneous pancreas-kidney transplantation. In chapter 4, acute rejection in kidney transplant recipients resulted in higher circulating LNC-EPHA6 levels. In chapter 5, clinical parameters and single antigen bead assay for measurement of donor specific antibodies were evaluated in the context of antibody-mediated rejection. Female recipients who received a kidney transplant from their spouse were especially at risk for acute antibody-mediated rejection and a single antigen bead assay is more sensitive to detect antibodies in this group than the standard diagnostic strategy. In chapter 6, mesenchymal stromal cell therapy is studied in a randomized controlled trial to evaluate the potential for prevention of acute rejection and fibrosis in transplanted kidneys. MSC therapy resulted in similar fibrosis scores and rejection rates. Show less
In recent years HLA epitope matching is becoming a hot topic in transplant community to prevent donor-specific antibody formation after transplantation, as such antibodies are associated with... Show moreIn recent years HLA epitope matching is becoming a hot topic in transplant community to prevent donor-specific antibody formation after transplantation, as such antibodies are associated with inferior graft survival. The number of HLA epitope mismatches between donor and recipient correlates with donor-specific antibody formation, but not every epitope mismatch will trigger an antibody response. Therefore, it is pivotal to define the immunogenic epitopes. In my thesis we describe the development of the software HLA-EMMA to determine HLA amino acid mismatches between donor and recipient to identify immunogenic amino acids. In addition, the generation of recombinant human HLA-DR monoclonal antibodies for antibody-verification of eplets/epitopes is described. These tools will contribute to the definition of immunogenic epitopes, which is required before introducing HLA epitope matching in the clinic. Show less
The aim of the research described in this thesis was to obtain more insight in the risk factors of BK polyomavirus (BKPyV) infection after kidney transplantation (KTx), with special emphasis on... Show moreThe aim of the research described in this thesis was to obtain more insight in the risk factors of BK polyomavirus (BKPyV) infection after kidney transplantation (KTx), with special emphasis on pretransplantation related risk factors. Both donor and recipient as well as viral factors, were investigated. The ultimate goal was to identify reliable predictive markers of BKPyV infection after KTx, thereby providing opportunities to optimize and personalize the currently recommended BKPyV screening strategy. Chapter Two describes the correlation between pretransplantation donor-recipient pair seroreactivity against BKPyV and development of BKPyV viremia and BKPyVAN after KTx. In Chapter Three the stability of BKPyV seroreactivity in KTRs and healthy blood donors, and the correlation of BKPyV seroreactivity with preceding viremia in KTRs is described. In Chapter Four the reduced risk of BKPyV infection in HLA-B51 positive recipients after KTx is described. Chapter Five describes the development and evaluation of a Luminex bead-based multiplex immunoassay for BKPyV serotyping. In Chapter Six the application of the Luminex bead-based multiplex immunoassay for BKPyV serotyping is described in a cohort of KTx donor-recipient pairs. In the General Discussion implications for prediction of BKPyV infections in recipients after KTx, as well as suggestions for further research are described. Show less
Increased patient involvement has been show to result in better clinical outcomes and increased autonomy and to offer a way to control volume and costs of chronic healthcare. The main research... Show moreIncreased patient involvement has been show to result in better clinical outcomes and increased autonomy and to offer a way to control volume and costs of chronic healthcare. The main research question of this thesis was whether part of the kidney transplant aftercare can be delegated to patients without loss of quality of care. The results of the studies described show that self-monitoring kidney function after transplantation is an attractive option to kidney transplant patients and can lead to a significant decrease in number of outpatient visits without compromising on quality of care. Further, self-monitoring could offer a relatively cheap way to increase monitoring frequency, which could lead to earlier detection and treatment of complications and, consequently, improved clinical outcomes. To unravel the full potential of self-monitoring kidney function after transplantation, it is recommended to use accurate measurement devices that both patients and healthcare professionals have confidence in, design a protocol with the involvement of a multidisciplinary group that is truly representative of all stakeholders (including patients and physicians that are more critical of self-monitoring) and choose a study design that includes formative evaluations instead of summative evaluations alone. Show less
Within this thesis several novel strategies to regenerate the human kidney are exploited. The first strategy is to improve kidney function is by mesenchymal stromal cell (MSC) therapy. In chapter 2... Show moreWithin this thesis several novel strategies to regenerate the human kidney are exploited. The first strategy is to improve kidney function is by mesenchymal stromal cell (MSC) therapy. In chapter 2 the current status of clinical trials with MSC therapy are discussed. In chapter 3 we show an extensive characterization of MSCs derived from human kidney (hkPSCs) compared to bone marrow derived MSCs (bmMSCs) and show that hkPSCs show organotypic expression signatures and functionality. For fluent clinical translation, we developed a clinical grade acceptable standard operation procedure (SOP) (chapter 4). In chapter 5 we show that the cytokine secretion profile of both hkPSCs and bmMSCs was closely related to cell morphology adaptation to culture surface topography and was stromal cell type specific. In chapter 6 we show that not only the kidney cortex but also the kidney capsule contains a stromal cell population. In chapter 7 we report the regeneration of kidney vasculature by repopulating the vascular compartment of human and rat kidney matrices with hiPSC-derived endothelial cells. We show efficient cell delivery, adherence and survival of these endothelial cells as a first, but critical, step towards a human bioengineered kidney. Show less
This thesis provides insights into the mechanisms of renal I/R injury based on human kidney transplantation (i.e. the status of delayed graft function: DGF). A severe energetic crisis... Show moreThis thesis provides insights into the mechanisms of renal I/R injury based on human kidney transplantation (i.e. the status of delayed graft function: DGF). A severe energetic crisis differentiates DGF kidneys from adequately functioning controls. Although intact beta-oxidation, aerobic glycolysis and glutaminolysis provide Krebs Cycle intermediates, these intermediates are not able to enter the mitochondrial Krebs cycle. Hence, dysfunctional mitochondria disable efficient ATP production leading to the metabolic incompetence that causes DGF and underlies renal I/R injury. This finding sheds a whole new light on I/R injury and explains why ATP-dependent therapeutics are ineffective as treatment for I/R injury. A major difference in the vulnerability of mitochondria to ischemia and reperfusion between rodents and humans was found. This could explain the current differences in effectiveness of therapies in the experimental versus the clinical setting. Big cohort studies give insights in donor, recipient and transplant-procedure variables and challenge the reluctance towards the use of DCD donor kidneys. New preventive strategies could limit I/R injury by preserving mitochondria (hypothetically with peptide SS-31 or activation of mitochondrial aldehyde dehydrogenase). This will overcome the detrimental effects of I/R injury on graft function and survival - thereby increasing the success rate of kidney transplantation. Show less
Acute kidney transplant rejection is an important risk factors for adverse graft outcome. Once diagnosed, it remains difficult to predict the risk of graft loss and the response to anti-rejection... Show moreAcute kidney transplant rejection is an important risk factors for adverse graft outcome. Once diagnosed, it remains difficult to predict the risk of graft loss and the response to anti-rejection treatment. The aim of this thesis was to identify biomarkers during acute rejection, which predict the response to corticosteroid therapy and renal allograft survival. We demonstrated that steroid resistance is a multifactorial condition, in which both immunological and non-immunological factors are involved. Response to steroid therapy correlates with the expression level and characteristics of allograft infiltrating T cells and macrophages, indicating that steroid resistance resides in specific cell populations and is not a feature of all lymphocytes. In addition, zinc regulation plays a role in the response to corticosteroids. Increased expression of zinc-regulating molecules may diminish the zinc-requiring anti-inflammatory effects of corticosteroids. Therefore, kidney transplant recipients may benefit from additional zinc intake to optimize steroid signaling. Furthermore, we demonstrated that a multivariate prediction model, containing biomarkers related to different aspects of corticosteroid signaling, offers the best prognostic value for assessing steroid response. Finally, we demonstrated that determination of S100A8 and S100A9 expression levels in renal allograft tissue can be used for assessing the risk of renal allograft loss over time. Show less
Calcineurin inhibitor (CNI)-based therapy is associated with nephrotoxicity and cardiovascular adverse effects in renal transplant recipients. Early CNI withdrawal with mycophenolate mofetil (MMF)... Show moreCalcineurin inhibitor (CNI)-based therapy is associated with nephrotoxicity and cardiovascular adverse effects in renal transplant recipients. Early CNI withdrawal with mycophenolate mofetil (MMF) has not become routine practice, due to concerns about acute rejection. Therapeutic drug monitoring (TDM) may be advantageous when a CNI or MMF is withdrawn. The impact of late concentration-controlled CNI withdrawal with MMF on renal function, the incidence of acute rejection and markers of cardiovascular disease was evaluated in a randomised trial. In 158 stable renal transplant recipients on a CNI-based regimen with prednisone and MMF either the CNI or MMF was withdrawn. A total of 119 patients participated in the cardiovascular substudy. Late concentration-controlled CNI withdrawal resulted in improved renal function, especially in patients with an estimated glomerular filtration rate of less than 50 ml/min/1.73 m2, with a low acute rejection rate. The progression of left ventricular diastolic dysfunction was prevented by CNI elimination. CNI withdrawal decreased ambulatory blood pressures, but had no specific impact on carotid IMT. In conclusion, late CNI withdrawal with TDM of MMF may result in improved outcome by beneficial effects on renal function and cardiovascular risk, with a low risk of rejection in the majority of stable renal transplant recipients. Show less
In this thesis, acute rejection after kidney, simultaneous pancreas and kidney (SPKT), and islets of Langerhans transplantation was addressed. The focus is on acute antibody-mediated rejection (AMR... Show moreIn this thesis, acute rejection after kidney, simultaneous pancreas and kidney (SPKT), and islets of Langerhans transplantation was addressed. The focus is on acute antibody-mediated rejection (AMR) after transplantation and on a potential strategy using cellular immune modulation to prevent acute rejection. First, we retrospectively evaluated the relevance of diffuse C4d-positive peritubular capillary staining in a well-defined kidney transplantation cohort with proven early acute rejections (Chapter 2). Second, the negative impact of AMR on pancreas graft survival was investigated, which proved to be significant (Chapter 3). We subsequently analyzed all SPKT patients at the LUMC with early pancreas graft loss to examine the role of AMR due to presumed thrombosis and/or acute rejection (Chapter 4). In Chapter 5, a clinical update is presented on islets of Langerhans transplantation, focusing on the alternative _-cell replacement therapy that is currently employed in the LUMC (Chapter 5). Furthermore, we reviewed the role of both lymphatic- and blood-vessel vascularization and the role of neuronal reconnection after islet transplantation with data from our own rat islet transplantation models (Chapter 6). Finally, in a rodent allogeneic islet transplantation model, we attempted to induce tolerance by using donor-derived, dexamethasone-pretreated dendritic cells (Chapter 7). Show less
Calcineurin inhibitors are crucial in the prevention of acute rejection in the first year after renal transplantation. Unfortunately, these drugs (ciclosporin A, tacrolimus) are characterized by... Show moreCalcineurin inhibitors are crucial in the prevention of acute rejection in the first year after renal transplantation. Unfortunately, these drugs (ciclosporin A, tacrolimus) are characterized by serious clinical toxicity and between patient variability in their effect. Therefore, the dose of these drugs should be individualized in order to reach a balance between rejection and toxicity. This thesis aimed to describe the variability between and within patients using mathematical models and subsequently to explain this variability. Genetic and non-genetic factors were used to explain variability and several factors were identified (polymorphism in metabolism enzyme CYP3A5, body weight, concomitant prednisolone dose). For this purpose drug concentrations in blood are measured as a concentration biomarker. Furthermore, another biomarker the activity ot the target enzyme calcineurin was determined in leukocytes, but was found to be more variable within patients than between patients. This response biomarker was not found to be clinically useful to individualize the drug dosage. Finally, pharmacological determinants for subclinical acute rejection at 6 months were determined in patients treated with ciclosporin. Although ciclosporin exposure and several genetic variants were not found to relate, a previous acute rejection period and a kidney from a deceased donor increased the risk of rejection 5-fold. Show less
The innate immune system plays an important role in solid organ transplantation. This thesis focuses on the role of the lectin pathway of complement activation in kidney and simultaneous pancreas... Show moreThe innate immune system plays an important role in solid organ transplantation. This thesis focuses on the role of the lectin pathway of complement activation in kidney and simultaneous pancreas-kidney transplantation (SPKT) and describes the role of properdin in tubular complement activation and clearance of apoptotic cells. Mannose-binding lectin (MBL) is the initiating molecule of the lectin pathway of complement activation and its levels are largely determined by frequently occurring polymorphisms of the MBL gene. Our studies show that high MBL levels in the transplant recipient are associated with poorer graft survival and in the case of SPKT also with poorer survival of the recipient. While low MBL levels are associated with better survival we also show that low MBL does increase the risk for infectious complications after SPKT. Properdin is classically described as a stabilizer of the alternative pathway of complement activation. This thesis points towards a new role for properdin by describing a direct interaction with apoptotic cells and with the tubular epithelium of the kidney. By directly binding to these cellular surfaces properdin may contribute to the clearance of apoptotic cells and participate in complement activation and renal damage in proteinuric states. Show less
Following allograft transplantation, the immune system is triggered to induce an immunogenic response against the non-self organ. To prevent the induction of this immunogenic response, recipients... Show moreFollowing allograft transplantation, the immune system is triggered to induce an immunogenic response against the non-self organ. To prevent the induction of this immunogenic response, recipients are treated with immunosuppressive medication. The majority of these medications target T cells, which play a key role in the rejection process, and thereby prevent acute rejection in most of the recipients. Non-specific targeting of these T cells not only prevents acute rejection, it also prevents responses against pathogens or tumor growth. In addition, long-term use of immunosuppressive agents may cause organ failure due to toxic effects on the organ [1]. Therefore, the ultimate goal is to develop a therapy, which targets alloreactive T cells, allowing a normal response against pathogens and tumors, in the absence of chronic use of immunosuppressive agents. Various strategies have been employed to induce such a donor-specific tolerance, amongst which treatment with immature DC [2]. These immature DC have, in contrast to mature DC, the capacity to induce tolerogenic responses and are therefore an attractive candidate for cellular therapy. The studies presented in this thesis demonstrate that in fully mismatched kidney transplantation models, administration of modulated donor-derived DC to recipient__s results in regulation of recipient__s immune response. Both the donor-specific hyporesponsiveness of recipient T cells and the reduced influx of CD8+ T cells into the graft of LPS-DexDC treated recipients indicate a positive effect of this treatment. However, optimization of this treatment is necessary, since no prolonged allograft survival was induced. Several mechanisms, which are not regulated by LPS-DexDC, may be responsible for the observed rejection, amongst which the preformed alloantibodies, increased levels of C3 in the graft and the increased influx of NK cells. Additional studies are required to explore the modulating effects of antibodies which block co-stimulation and/or short courses of immunosuppressive drugs as a co-treatment in these settings. Show less
