Pharmaceutical companies, governments and the general public have become increasingly aware that animal models used in drug testing lack vital aspects to serve as an accurate representation of... Show morePharmaceutical companies, governments and the general public have become increasingly aware that animal models used in drug testing lack vital aspects to serve as an accurate representation of human biology. As models of the human body should become more physiologically relevant, animal models no longer suffice because responses of animal cells often differ from human cells. Instead, in vitro cell culture models with 3D architecture, microfluidics and high throughput capabilities are a promising technology. These 3D models can be developed in ways that they will likely surpass animal models on important aspects like resemblance to a human body, predicting compound safety and efficacy, high throughput testing capabilities, ethical aspects, and costs.To demonstrate the feasibility of an advanced 3D in vitro model, we used a microfluidic in vitro platform to develop a kidney-on-a-chip platform which possess the ability to reproduce the tubular response to known and unknown nephrotoxicants and compounds as seen in in vitro and in clinical studies. Furthermore, we assessed the response of the model to renal ischemia/reperfusion injury and could measure the prevention of tubular damage when adding protective compounds.These findings show that 3D tissue models can compete with alternatives like animal models and 2D models. Show less
The aim of the research described in this thesis was to study the role of complement in renal ischemia/reperfusion injury (IRI) and to delineate the contribution of the different complement... Show moreThe aim of the research described in this thesis was to study the role of complement in renal ischemia/reperfusion injury (IRI) and to delineate the contribution of the different complement pathways involved. So far, in human renal IRI, the activation pathways of complement by ischemic proximal tubular epithelial cells (PTEC) are still incompletely elucidated. In chapter 2 we therefore established an in vitro model to simulate IRI on human and mouse PTEC by culturing these cells under normoxic or hypoxic conditions and then investigated the subsequent effects on complement activation following reoxygenation (reperfusion). We specifically focused on the question which pathway(s) of complement activation are initiated by human and mouse PTEC after hypoxic stress. In chapter 3 we addressed the lack of evidence for involvement of complement in human IRI. We assessed the formation and release of C5b-9 during early reperfusion in clinical kidney transplantation in living donor, brain-dead donor, and cardiac dead donor kidney transplantation. Complement activation following I/R may take place in both tubular and vascular compartments. Therefore, we systematically measured terminal complement activation during early reperfusion in human kidney transplantation in both the tubular compartment by immunohistochemistry and the intravascular compartment by selective arteriovenous measurements over the transplanted kidney. In chapter 4 we studied whether C5b-9 could also be detected in urines of transplant recipients early after transplantation. In addition, we investigated the possibility whether in proteinuric urine, which is common following transplantation, C5b-9 might be generated independent of a renal contribution. Based on previous clinical studies that high serum levels of MBL were associated with inferior renal allograft survival following clinical transplantation, we examined in chapter 5 the role of MBL in the pathophysiology of renal IRI and explored the therapeutic targeting of MBL in a rat model of renal IRI. We identified an entirely novel role for MBL in mediating reperfusion-induced kidney injury following ischemia which is completely independent of complement activation. In chapter 6 we studied the mechanism by which MBL might mediate tubular injury following renal I/R. Vascular leakage results in exposure of tubular cells to MBL, which was shown to be the primary culprit of tubular injury. Recombinant human C1 inhibitor (rhC1INH) is a serine protease inhibitor that inhibits complement activation, reduces vascular permeability and interacts with MBL. In chapter 7 we therefore explored the therapeutic application of rhC1INH in renal IRI and studied whether rhC1INH is able to attenuated MBL-mediated kidney injury. In chapter 8 we investigated the impact of short- and long-term IRI on vascular integrity, pericytes and angiopoietin expression. Finally, in chapter 9 the findings presented in this thesis are critically discussed and the possible implications for kidney transplantation are presented Show less
