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