Chronic kidney disease is a major global public health issue. It involves progressive nephron loss, characterized by alterations in the structure and function of the kidneys, due to sustained injury. When chronic kidney disease advances to end-stage kidney disease, renal replacement therapy (dialysis or kidney transplantation) remains the only effective therapeutic intervention. Given the increasing incidence of chronic kidney disease, its irreversible nature, aging populations, and limited availability of effective treatments, a deeper understanding of its underlying mechanisms and the development of novel therapeutic approaches are of great public and scientific interest. Animal studies raise ethical and translational concerns, while conventional cell culture models fail to accurately represent complex intercellular interactions and communication. Therefore, there is an urgent need for advanced research models capable of reproducing a heterogeneous three-dimensional (3D) kidney microenvironment. Two distinct human cellular models were developed to examine different aspects of the glomerulus: cell interactions and the glomerular filtration barrier, focusing on how co-cultivation and flow conditions influence model maturation. Human primary glomerular endothelial cells, conditionally immortalized podocytes, and primary mesangial cells were cultured in 2D or as spheroids, either in monoculture or as co-culture. 3D glomerular co-cultures self-organized into spheroids with specific cell arrangements, exhibited improved cell viability and extracellular matrix production compared to monocultured spheroids, based on a Live/Dead assay and immunofluorescent analyses, respectively. Notably, when comparing 2D cultures with corresponding 3D cultures, transcriptional analyses, including bulk RNA sequencing and qPCR, confirmed a significant upregulation of cell-specific markers, extracellular matrix-related genes, and genes involved in cell attachment and cross-talk. Importantly, housekeeping genes must be carefully selected, as many commonly used genes are affected by 3D culture conditions. Communication between podocytes and glomerular endothelial cells was further examined by tracking podocyte-derived VEGF-A to glomerular endothelial cells. Despite the described benefits of incorporating fiber into spheroids for reducing hypoxia, our study found no effects of poly-L-lactic acid or gelatin fiber incorporation on the expression of hypoxia-target genes. At the same time, co-culturing with fibers caused an undesirable, significant decrease in the expression of the podocyte-specific marker SYNPO, thereby hindering our model of highly differentiated cells. The glomerular filtration barrier, comprising glomerular endothelial cells, podocytes, and the glomerular basement membrane, is fundamental to the kidney’s selective filtration function. Structural or functional impairment of any component of the glomerular filtration barrier disrupts filtration integrity, resulting in proteinuria and progressive renal failure. In order to replicate critical features of the native glomerular filtration barrier, a poly-L-lactic acid-based electrospun membrane was used as an artificial glomerular basement membrane, providing a biocompatible scaffold for glomerular endothelial cells and podocytes. The biomimetic setup supported monolayer formation, specific marker expression, and proper morphology of both cell types. Importantly, the fiber topography of the membrane influenced cell shape and layer integrity, as shown by scanning electron microscopy. The model's functionality was evaluated by testing barrier leakiness with 10 kDa and 70 kDa dextrans. The colonized artificial glomerular filtration barrier prevented the passage of 70 kDa dextran, confirming its selectivity. Meanwhile, 10 kDa dextran and other small molecules, such as VEGF-A and miRNAs, could cross the barrier, enabling bidirectional glomerular communication. Given the influence of fluid shear stress on glomerular endothelial cell maturation and fenestrae development, flow conditions were implemented in a custom 3D-printed micro-bioreactor. Subsequent imaging with scanning electron microscopy and gallium-focused ion beam technique revealed the development of fenestrae on the glomerular endothelial cell surface. Additionally, the system was evaluated for compatibility with human induced pluripotent stem cell- derived podocytes, paving the way for studies involving patient-derived mutation-carrying cells and personalized medicine. In conclusion, two advanced human cellular models were established. The results obtained in this thesis suggest an essential role of co-culturing, 3D microenvironment, and flow conditions for cell maturation. By mimicking cross-cell communication or structural architecture, current models represent valuable tools for basic kidney research as well as disease modeling, and can be used to evaluate promising candidates for interventional therapies.
Anna Rederer (Thu,) studied this question.