Generation of human glomerular spheroids and filtration barrier in a vascularized milieu as a personolized model for glomerular diseases

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Within the kidney, the glomerulus is the functional unit where the urine is filtered from the blood through the glomerular filtration barrier. This barrier consists of fenestrated glomerular endothelial cells, the glomerular basement membrane and podocytes. Impairments in podocytes can lead to nephrotic syndrome. Mutations in podocyte gens is a cause for steroid resistant nephrotic syndrome (SRNS). In autosomal dominant mutations, the disease usually manifest in young adulthood suggesting a second hit. SRNS is believed to not response to immunosuppressive therapy and to progress to end stage renal disease. However, recent data suggest that a combination of cyclosporine A and steroids can lead to remission in some cases. Mechanisms of this therapeutic concept are largely unknown. For years, cell culture models have been used to study glomerular diseases ex vivo. However, these models are limited in their resemblance to nature glomeruli due to lack of 3-D context, glomerular crosstalk between different cell types, absence of patient derived cells, and a vascularized milieu. This project aims to establish glomerular structures to better understand glomerular functions and interaction under physiological and pathophysiological conditions. Therefore, we will generate glomerular spheroids with different personalized iPSC derived human podocytes, human glomerular endothelial cells and mesangial cells in co-culture ex vivo and later vascularize these structures in our established AV loop rat model.Furthermore, we will culture patient-derived podocytes together with human glomerular endothelial cells on two sites of an artificial basement membrane and vascularize this artificial basement membrane in our AV loop rat model. To generate iPSC podocytes of patients we will use skin fibroblast of patients with mutations in WT1 and INF2 as well as healthy volunteers, reprogram them into iPSCs and differentiate them into podocytes keeping the patients mutation. Finally, we will explant the vascularized models and use them for different therapeutic and toxic screenings. Single cell seq analysis and morphological characterization of the vascularized co-culture models will allow analyzing the patient derived podocytes in the context of other glomerular cells with and without treatment. Herewith, we want to test therapeutic options in SRNS as well as susceptibility towards toxic substances in a patient-individualized manner.