Diabetes mellitus and its complications stands as arguably the most formidable pandemic of the 21st century. While rodent models of diabetes mellitus have been extensively explored, none have managed to faithfully replicate the full spectrum of pathological hallmarks and secondary complications witnessed in diabetic patients. Among the commonly affected organs is the kidney, manifesting in the form of diabetic kidney disease (DKD). Recently, our clinical understanding of incretins as critical regulators of disease progression in diabetic patients including DKD has undergone significant expansion. In particular, the incretin hormone gastric inhibitory polypeptide (GIP) axis has taken central stage. A ground-breaking development in this realm was the creation of a GIP receptor dominant negative (GIPRdn) mouse, exhibiting all the characteristic features observed in DKD patients. This study sheds light on the heightened susceptibility of these mice to lethal acute kidney injury (AKI) induced by ischemia-reperfusion injury (IRI). Notably, isolated renal GIPRdn-tubules displayed accelerated cell death propagation and increased tubular necrosis. Expanding on previous cell culture experiments involving hyperglycemia, it became apparent that tubules of GIPRdn mice express elevated levels of the intracellular thioredoxin interacting protein (TXNIP), previously reported to be responsible for the degradation of glucose transporter 1 (GLUT1). This phenomenon is crucial in maintaining intracellular glucose homeostasis. The study further indicates an association between TXNIP and the downregulation of thioredoxin reductase 1 (TXNRD1), a selenoenzyme playing a pivotal role in protecting renal tubules from ferroptosis in a glutathione-independent manner. Intriguingly, the inhibition of TXNRD1 with the small molecule ferroptocide (FTC) in GIPRdn tubules resulted in severe tubular necrosis, a condition effectively reversed by the ferroptosis inhibitor ferrostatin 1 (Fer-1). This nuanced exploration establishes a connection between DKD and a heightened sensitivity to kidney tubular ferroptosis, thereby presenting a potential avenue for intervention with ferrostatins. Importantly, the administration of a single dose of Fer-1 significantly prolonged the survival of GIPRdn mice following IRI. In conclusion, this study illuminates the intricate dynamics of DKD, highlighting a pronounced sensitization to kidney tubular ferroptosis. The findings suggest that ferrostatins, particularly exemplified by Fer-1, hold promise as potential therapeutic agents in mitigating the severity of this condition, offering hope for improved outcomes in individuals struggling with diabetes-related kidney complications.:Acknowledgments Abstract Zusammenfassung List of abbreviations List of tables List of Figures 1. Introduction 1.1. Diabetes mellitus 1.1.1. Definition and description 1.1.2. Epidemiology 1.1.3. Classification of diabetes mellitus 1.1.4. Diagnosis of diabetes mellitus 1.1.5. Type 2 Diabetes Mellitus 1.1.6. Long-term complications of T2DM 1.1.6.1. Diabetic Nephropathy 1.1.6.2. Therapies for diabetic nephropathy 1.1.7. Animal models for diabetic kidney disease 1.1.7.1. Diabetic eNOS knockout mouse 1.1.7.2. Bradykinin B2 Receptor (B2R) deficient Ins2Akita/+ mouse 1.1.7.3. Decorin-deficient streptozotocin diabetic mouse 1.1.7.4. NONcNZO mouse 1.1.7.5. OVE26 mouse 1.1.7.6. Black and tan, brachyuric (BTBR) ob/ob mouse 1.1.8. Incretin hormones and GIPRdn diabetic mouse model 1.1.8.1. Generation of GIPRdn diabetic mouse model 1.2. Regulated cell death 1.3. Ferroptosis 1.3.1 Mechanism of ferroptosis 1.3.1.1 Sensitization to ferroptosis by ether phospholipids 1.3.1.2 Hydropersulfides and ferroptosis 1.3.2 Ferroptosis inducers (FINs) and inhibitors 1.3.3 Ferroptosis in the kidney 1.4 Aims 2. Materials and Methods 2.1. Reagents 2.2. Experimental models: cell lines and mouse strains 2.2.1. Cell culture conditions 2.2.2. Mice 2.2.2.1. Genotyping 2.2.2.1.1. DNA isolation 2.2.2.1.2. Polymerase Chain Reaction (PCR) 2.2.2.1.3. Gel electrophoresis 2.2.2.2. Body weight 2.2.2.3. Blood glucose 2.2.2.4. Blood collection and serum parameters 2.2.3. Isolation of primary murine renal tubules 2.2.4. Generation of a 3D-printed double chamber 2.3. Experimental procedures 2.3.1. Plating and treatment of cells 2.3.2. Fluorescence activated cell sorting (FACS) 2.3.3. Western Blotting (WB) 2.3.4. Induction of cell death on isolated murine tubules 2.3.5. LDH release assay 2.3.6. Evaluation of speed of cell death propagation (exponential plateau – growth equation) 2.3.7. Time lapse imaging and processing of the time lapse data 2.3.8. Fluorescence Lifetime Imaging Microscopy (FLIM) 2.3.8.1. Time domain data analysis 2.3.8.2. FLIM time lapse video generation 2.3.9. Thioredoxin Reductase Activity assay 2.3.10. Bilateral kidney Ischemia and Reperfusion injury (IRI) 2.3.11. Immunohistology and semi-quantitative scoring 2.3.12. Measurements of sulfur-containing metabolites by ultra-performance liquid chromatography-mass spectroscopy (LC-MS) 2.4. Statistical analysis 3. Results 3.1. Characterization of diabetic kidney disease in GIPRdn mice 3.1.1. Blood glucose viii 3.1.2. Body weight 3.1.3. Serum parameters 3.1.4. Histological analysis of the kidneys 3.2. The spontaneous death of GIPRdn tubules is characterized by a non-random pattern of necrotic cell death 3.3. GIPRdn tubules are more prone to undergo spontaneous death compared to WT tubules 3.4. Spontaneous necrosis of GIPRdn and WT tubules is partially mediated by ferroptosis 3.5. GIPRdn tubules show downregulation of the PRX pathway compared to the non-diabetic tubules 3.6. GIPRdn tubules show altered hydropersulfides pathway 3.7. GIPRdn tubules show altered etherglycerophospholipids (etherPLs) pathway. 3.8. Ferrostatin-1 but not Empagliflozin reverses ferroptosis induction in different cell lines as well as in isolated kidney tubules 3.9. GIPRdn mice are more sensitive to IRI-induced acute kidney injury compared to their WT littermates 3.10. Ferrostatin-1 ameliorates the sensitivity of GIPRdn to ischemia reperfusion injury-induced acute kidney injury 4. Discussion 4.1. The GIPRdn mouse model 4.2 Ferroptosis in diabetic nephropathy 4.2.1. Ferroptotic cell death is involved in the spontaneous death of diabetic tubules 4.2.2. Possible mechanisms behind the enhanced sensitivity of the GIPRdn kidney tubules to ferroptosis 4.3. Therapeutic consequences of the study 4.3.1. SGLT2 inhibitor empagliflozin does not have a protective effect on diabetic tubules undergoing spontaneous death 4.4. Outlook and limitations of the study References

Die Homöostase wird maßgeblich durch die Niere, bestehend aus Millionen funktioneller Untereinheiten, den Nephronen, aufrechtherhalten. Chronisch geschädigte Nephrone führen zur Entwicklung einer terminalen Nierenerkrankung (TNE). Die Erzeugung renaler Zellen aus humanen pluripotenten Stammzellen (hPSCs) stellt eine vielversprechende Strategie zur regenerativen Therapie und Behandlung von TNE dar. In der vorliegenden Arbeit wurde ein Protokoll zur Differenzierung von renalen Vorläufern (RV) aus hPSCs entwickelt, welches nephronale Zelltypen und Strukturen in vitro und ex vivo erzeugte. Eine selektierte Kombination von Faktoren wurde in diesem 8-Tage-Protokoll genutzt, um die schrittweise Differenzierung der hPSCs zu lenken, indem die embryonale Organogenese der Niere abgebildet wurde. Am Tag 6 der Differenzierung konnten SIX2+/CITED1+ Zellen des metanephrischen Mesenchyms und HOXB7+/GRHL2+ Zellen, welche auf Vorläufer der Ureterknospe hindeuten, nachgewiesen werden. Diese entwickelten sich am Tag 8 weiter zu LGR5+/JAG1+/WT1+ renalen Vesikelzellen. Weiterführende Kultivierung in drei verschiedenen induktiven Medien führte zu WT1+/PODXL+/SYNPO+ Podozytenvorläufern, PDGFRß+/DESMIN+/αSMA+ Mesangialzellen und epithelialen Zellen des proximalen und distalen Tubulus sowie des Sammelrohrs. Außerdem bildeten die Tag-8-Vorläuferzellen spontan 3D renale Organoide aus. Die RV induzierten tubuläre Strukturen an einer Luft-Flüssigkeits-Grenzfläche und integrierten sich in embryonale Nierenaggregate. Zusammenfassend konnte demnach ein Protokoll entwickelt werden, welches entstehenden Nephronen ähnliche RV generierte, die innerhalb von 14 Tagen in spezialisierte nephronale Zelltypen differenzierten. Diese einfache Methode, um renale Zellen aus einem gemeinsamen Vorläuferpool in einer 2D -Kultur zu erzeugen, schafft die Grundlage für eine Produktion im größeren Maßstab, sowie für Modellsysteme in toxikologischen Untersuchungen oder Zelltherapien. / Kidneys are the central organ for homeostasis for our body systems and composed of around a million functional units, the nephrons. Chronically damaged nephrons deteriorate progressively towards end stage renal disease (ESRD), owing to the limited regenerative capacity of adult mammalian kidneys. The generation of renal cells from human pluripotent stem cells (hPSCs) is a promising strategy to develop regenerative therapies for ESRD. In this study, we established a protocol to differentiate hPSCs to renal progenitors (RP), capable of producing nephronal cell types and structures in vitro and ex vivo. An effective combination of factors obtained after intensive screening, was used to create an 8-day-protocol that steered hPSCs to the renal lineage by a step-wise process outlining the embryonic milestones in kidney organogenesis. Six days after growth factor treatment, a mixture of SIX2+/CITED1+ cells representing metanephric mesenchyme and an HOXB7+/GRHL2+ population indicative of ureteric bud progenitors was obtained that developed into LGR5+/JAG1+/WT1+ renal vesicle cells by the day 8. Prolonged cultivation of these day 8 cells in three inductive media resulted in generation of WT1+/PODXL+/SYNPO+ podocyte-precursors, PDGFRß+/DESMIN+/αSMA+-mesangial cells and fractions of proximal, distal and collecting duct tubular epithelial cells in vitro. Moreover, day 8 cells differentiate spontaneously into renal organoids in culture. The hPSC-derived RP gave rise to tubular structures upon culture as a pellet in air-liquid interface and integrated into embryonic kidney re-aggregations. Thus, we demonstrate that our protocol generates RP reminiscent of nascent nephrons, which can be coaxed into specialized nephronal cell types in vitro after 14 days from hPSCs. This simple and rapid method to produce renal cells from a common precursor pool in 2D culture provides the basis for scaled-up production of tailored renal cell types, applicable for drug testing or cell therapies.

Renal tubules represent an intercellular unit and function as a syncytium. When acute tubular necrosis was first visualized to occur through a process of synchronized regulated necrosis (SRN) in handpicked primary renal tubules, it became obvious that SRN actually promotes nephron loss. This realization adds to our current understanding of acute kidney injury (AKI)-chronic kidney disease (CKD) transition and argues for the prevention of AKI episodes to prevent CKD progression. Because SRN is triggered by necroptosis and executed by ferroptosis, 2 recently identified signaling pathways of regulated necrosis, a combination therapy employing necrostatins and ferrostatins may be beneficial for protection against nephron loss. Clinical trials in AKI and during the process of kidney transplantation are now required to prevent SRN. Additionally, necrotic cell death drives autoimmunity and necroinflammation and therefore represents a therapeutic target even for the prevention of antibody-mediated rejection of allografts years after the transplantation process.

Organoide aus adulten Mäusen sind vielversprechende Modelle für die Nierenforschung. Ihre Charakterisierung wurde jedoch nicht auf ein zufriedenstellendes Niveau gebracht. Hier habe ich ein langfristiges 3D-Maus-Organoid (Tubuloid)-Modell etabliert und charakterisiert, das die Erneuerung und die Reparatur sowie die Architektur und die Funktionalität der adulten tubulären Epithelien rekapituliert. In der Zukunft wird das Modell detaillierte Untersuchungen der Trajektorien selbsterneuernder Zellen sowohl zur teilweisen Wiederherstellung der Niere als auch zur malignen Transformation der Niere ermöglichen. Das klarzellige Nierenzellkarzinom (ccRCC) ist der häufigste und aggressivste Nierenkrebs. Die Inaktivierung des Tumorsuppressorgens Von Hippel-Lindau (VHL) ist der Haupttreiber des ccRCCs. Zuvor hatten wir die Hochregulation der Wnt- und Notch-Signalübertragung in den CXCR4+MET+CD44+-Krebsstammzellen (CSC) aus primären humanen ccRCC-Tumoren identifiziert. Das Blockieren von Wnt und Notch in von Patienten stammenden Xenotransplantaten, Organoiden und nicht-anhaftenden Sphären unter Verwendung von niedermolekularen Inhibitoren beeinträchtigte die Selbsterneuerung der CSC und das Tumorwachstum. Um CSC-gesteuertes humanes ccRCC in genetischen Mausmodellen nachzuahmen, begann ich mit der Erzeugung von zwei Doppelmausmutanten; β-Catenin-GOF; Notch-GOF und Vhl-LOF; β-Catenin-GOF. Sowohl die β-Catenin-GOF; Notch-GOF Mausmutante als auch die Vhl-LOF; β-Catenin-GOF Mausmutante entwickelten innerhalb einiger Monate schwere Krankheitssymptome. Überraschenderweise beobachtete ich weder Tumore oder Tumorvorläuferläsionen noch höhere Zellproliferationsraten in den mutierten Nieren. Weitere Analysen ergaben, dass die Mausmutanten Merkmale chronischer Nierenerkrankung (CKD) aufwiesen. / Adult mouse organoids are promising models for kidney research. However, their characterization has not been pushed forward to a satisfying level. Here, I have generated and characterized a long-term 3D mouse organoid (tubuloid) model, which recapitulates renewal and repair, and the architecture and functionality of the adult tubular epithelia. In the future, the model will allow detailed investigations of trajectories of self-renewing cells towards both the partial recreation and malignant transformation of the kidney. Clear cell renal cell carcinoma (ccRCC) is the most common and aggressive kidney cancer. Inactivation of the Von Hippel-Lindau (VHL) tumor suppressor gene is the major driver of ccRCC. Earlier, we identified the upregulation of Wnt and Notch signaling in CXCR4+MET+CD44+ cancer stem cells (CSCs) from primary human ccRCCs. Blocking Wnt and Notch in patient-derived xenografts, organoids and non-adherent spheres using small-molecule inhibitors impaired self-renewal of CSCs and tumor growth. To mimic CSC-governed human ccRCC in genetic mouse models, I started from the generation of two double mouse mutants; β-catenin-GOF; Notch-GOF and Vhl-LOF; β-catenin-GOF. Surprizingly, I observed neither tumors or tumor precursor lesions nor higher cell proliferation rates in the mutant kidneys. Further analyses revealed that the mutant mice displayed features of chronic kidney disease (CKD). Thus, β-catenin-GOF; Notch-GOF and Vhl-LOF; β-catenin-GOF mouse mutants did not develop kidney tumors under the given experimental conditions.