The prevalence of diabetes is sufficiently high to be classified as an epidemic, and 20-40% of these patients are expected to develop diabetic nephropathy, a leading cause of end-stage renal failure. Studies have identified a correlation between diabetic nephropathy and hypoxia in renal tissue in human studies. Increased oxygen consumption has been associated with the proton transport protein, uncoupling protein-2 (UCP-2), which uncouples the mitochondria. Previous research has reported elevated levels of UCP-2 in diabetic renal tissue. Consequently, it is crucial to determine how different preservation methods affect the detectability of UCP-2 in renal tissue for clinical applications. This study aimed to evaluate the effectiveness of Western blotting for detecting UCP-2 in snap frozen, fresh untreated, formalin-fixed, methyl carnoy-fixed, and RNA later-preserved rat kidneys. Preliminary trials were conducted to identify the optimal antibody combinations, followed by testing on various preserved tissues. The antibodies produced non-reproducible, unspecific, and unselective results. Additionally, technical challenges, such as gels adhering to membranes and low protein concentrations in some samples, rendered the results inconclusive. Further investigations are necessary to explore additional antibodies and variables that may influence the detection of UCP-2 in differently preserved tissues. Overall, this study highlights the complexity and challenges in developing reliable protocols for UCP-2 detection in preserved renal tissue, indicating that significant optimization is still required for consistent results.

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

Despite medical advances in the last decades, diabetic kidney disease (DKD) remains a major therapeutic challenge. DKD, the major microvascular complication in diabetic patients, is the most common cause of chronic kidney disease (CKD) and end-stage kidney disease (ESKD) requiring dialysis worldwide. In recent years, new therapeutic approaches for the treatment of DKD in addition to stringent blood glucose control and inhibition of angiotensin-signaling have been established. These new therapeutic options include sodium glucose cotransporter-2 inhibitors (SGLT2i), GLP-1 agonists, and nonsteroidal mineralocorticoid receptor antagonists. Despite their promising positive outcomes regarding kidney function, their long-term effects through the course of the disease remain unknown. Additionally, recent data show that about 50% of patients with DKD do not respond to these new therapeutics even if given in combination and there is a lack of therapies that can reverse the already established DKD. The continuous progression of diabetic complications (including DKD) despite normalization of blood glucose levels is referred to as the hyperglycemic memory. This phenomenon represents an unsolved medical problem in the field of diabetes management and remains without specific treatment options. Several theories have been proposed to explain the persistence of hyperglycemia-induced cellular dysfunction despite blood glucose normalization, and epigenetic regulation of gene expression has been proposed as the key pathomechanism driving the hyperglycemic memory. In this study, we used a combination of animal models, analyanalysesuman tissue and biofluid samples, in addition to in vitro mechanistic studies to identify therapeutically targetable pathways for the hyperglycemic memory in the context of DKD. To experimentally address the hyperglycemic memory, we used a model of hyperglycemia reversal in type-1 (STZ) and type-2 (db/db) diabetic mice. Hyperglycemia was reversed using SGLT2i (STZ and db/db mice) or insulin (STZ mice). We started by characterizing the functional and histological changes associated with hyperglycemic memory including persistent albuminuria, tubular hypertrophy, and fibrosis. To identify the pathways associated with the hyperglycemic memory in both models, we conducted bulk RNA sequencing from the kidneys and identified a large number of genes that were persistently up- or downregulated despite blood glucose normalization and hence possibly contributing to the hyperglycemic memory. Among these genes, we identified the cyclin-dependent kinase inhibitor p21 (Cdkn1a), which is known to regulate cellular senescence, as a primary candidate associated with the hyperglycemic memory, where its expression remained persistently upregulated despite blood glucose lowering in both diabetes models. Further investigations confirmed the “memorized” expression of p21 across DKD animal models and in cell lines in vitro, pinpointing the tubular epithelium as the specific cell type where this phenomenon occurs. Using a multimarker approach, we identified a persistent tubular senescence phenotype associated with p21 induction regardless of the intervention to reduce blood glucose levels in murine DKD models. Subsequent analyses aimed to scrutinize the relevance of this finding in the context of human DKD. We found induction of tubular p21 expression and senescence in human DKD biopsies compared to controls or to diabetic patients without kidney dysfunction. Furthermore, p21 was readily detected in the urine of DKD patients in a large cross-sectional cohort whic,h was increased with the severity of the disease and was negatively correlated with kidney function. Interestingly, urinary p21 levels remained persistently elevated despite different interventions to reduce blood glucose levels (SGLT2i or fasting-mimicking diet), corroborating its utility as a biomarker for the hyperglycemic memory in DKD. Next, we further elucidated the sequence of eventeventshyperglycemia to the induction of p21 and subsequent kidney damage, demonstrating that elevated blood glucose decreases DNA methyltransferase 1 (DNMT1) expression, resulting in hypomethylation of the p21 promoter and increased p21 expression. The induction of p21 expression triggers a senescence phenotype, that contributes to tubular damage and fibrosis. Suppression of tubular DNMT1 expression was sufficient to induce p21 in tubular cells, corroborating the importance of this epigenetic mechanism for the glucose-induced persistence of p21 expression. In order to investigate possible translational implications, we studied the potential of reversing the hyperglycemic memory in DKD. We employed the cytoprotective protease activated protein C (aPC), a disease resolving mediator associated with DKD protection. aPC in conjunction with the blood glucose lowering drug SGLT2i induced the expression of DNMT1 leading to promoter remethylation and suppression of the persistent tubular p21 expression. Notably, SGLT2i alone had no effect on p21 expression. The combined action of SGLT2i and aPC effectively counteracted albuminuria, tubular damage, senescence, and fibrosis in a murine DKD model. The protective effects of aPC were confirmed by investigating TMPro/Pro mice, in which thrombomodulin-mediated protein C activation is hampered resulting in reduced aPC levels. Induction of persistent hyperglycemia in these mice elevated p21 expression in association with aggravated kidney damage compared to wild-type mice. Superimposed deficiency of p21 in the aforementioned aPC-deficient mice (TMPro/Pro x p21-/-) alleviated the tubular injury and senescence phenotype, providing experimental in vivo evidence for a regulation of the hyperglycemic memory in DKD by the interaction of p21 by aPC levels. To confirm that aPC regulates DNMT1 and hence p21 in an epigenetic manner, we targeted DNMT1 in aPC-treated mice using the pan DNMT inhibitor 5-aza-2'-deoxycytidine or a specific vivo morpholino against DNMT1. Both approaches abolished aPC’s protective effects in a murine DKD model and inhibited its ability to reduce p21 promoter hypomethylation and hence its expression. Exploiting the cytoprotective properties of aPC by using the mutant 3K3A-aPC, which lacks the anticoagulant ability, or the chemical compound parmodulin-2 that mimics the biased signaling of aPC via the G protein-coupled receptor PAR1, was enough to reduce the hyperglycemia-induced p21 expression and the associated tubular senescence in murine DKD. The findings of this study uncover an important role of p21 in exacerbating renal damage under diabetic conditions, suggesting that p21 not only serves as a marker of cellular senescence but actively contributes to the persistence of DKD by mediating the hyperglycemic memory. By addressing the root causes of hyperglycemic memory, such as the epigenetic modifications that perpetuate p21 expression, it may be possible to halt or even reverse the progression of DKD. The feasibility of this approach was demonstrated by exploiting cytoprotective aPC signaling, which restored DNMT1 expression and reduced p21 expression. Thus, targeting the hyperglycemic memory in DKD may be feasible in general and may be specifically achieved by targeting cytoprotective aPC signaling.:Table of contents Table of contents 2 List of figures 5 List of tables 6 List of abbreviations 7 1. Introduction 9 1.1 Diabetes mellitus 9 1.2 Diabetic complications 9 1.3 Diabetic kidney disease (DKD) 10 1.3.1 Clinical presentation and staging of DKD patients 10 1.3.2 Cellular dysfunction in DKD 12 1.4 The hyperglycemic memory: a new challenge in DM management 14 1.4.1 Hyperglycemic memory in DKD 14 1.4.2 Mechanisms of the hyperglycemic memory 18 1.5 Cellular senescence in DKD 20 1.5.1 Features of senescent cells in DKD 20 1.5.2 Tubular cell senescence in DKD 23 1.6 Therapeutic management of DKD 23 1.6.1 SGLT2 inhibitors 24 1.6.2 Other therapeutic options for DKD 25 1.7 Coagulation proteases and their receptors in DKD 26 1.7.1 Protease-activated receptors (PARs) 26 1.7.2 Activated protein C (aPC) 27 2. Aim of the study 32 3. Methods 33 3.1 Reagents 33 3.2 Mice and in vivo interventions 34 3.3 Cell culture and in vitro interventions 35 3.4 Human renal biopsies and urine samples 36 3.5 Glucose uptake assay 41 3.6 In vitro Knockdown 41 3.7 Preparation of activated protein C 42 3.8 Urine collection and processing 42 3.9 p21 ELISA for human urine samples 43 3.10 Albuminuria and in mouse urine samples 43 3.11 Methylation specific PCR (MSP) 43 3.12 Pyrosequencing 44 3.13 DNMT activity assay 44 3.14 Immunoblotting 45 3.15 Reverse transcriptase PCR (RT-PCR) 45 3.16 Quantitative real time PCR (qRT-PCR) 46 3.17 RNA expression profiling 48 3.18 Functional annotation and Pathway analysis 48 3.19 Histology, immunohistochemistry and histological analyses 49 3.20 Immunofluorescence 49 3.21 Senescence associated beta galactosidase (SA-β-gal.) staining 50 3.22 Plasma creatinine and blood urea nitrogen (BUN) 50 3.23 Cell cycle analysis 50 3.24 Statistical Analysis 51 4. Results 52 4.1 Blood glucose normalization does not reverse DKD in experimental models of DM 52 4.2 Identification of genes and pathways associated with hyperglycemic memory 54 4.3 Sustained renal tubular p21 induction in experimental DKD models despite blood glucose normalization 56 4.4 Tubular p21 induction is independent on glomerular damage 58 4.5 Sustained renal tubular p21 expression is associated with induction of senescence 59 4.6. Induction of renal tubular p21 expression in human DKD patients is associated with kidney dysfunction 61 4.7. Sustained p21 expression despite glucose normalization in human DKD patients 63 4.8 aPC reverses glucose induced p21 promoter methylation and sustained p21 expression 64 4.9 Impaired protein C activation increases tubular p21 expression and senescence in vivo 66 4.10 p21 mediates enhanced tubular senescence in aPC-deficient mice 68 4.11 High glucose differentially regulates renal DNMTs expression and activity 70 4.12 Hyperglycemia-induced DNMT1 suppression is part of the hyperglycemic memory 72 4.13 aPC reverses glucose-induced and sustained renal p21 expression via DNMT1 in vivo 74 4.14 aPC reverses glucose-induced and sustained renal tubular senescence via DNMT1 in vivo 76 4.15 aPC requires PAR1 and EPCR to regulate p21 expression 78 4.16 aPC regulates the epigenetically sustained p21 expression independent of its anticoagulant function in vivo 79 4.17 aPC enhances the regenerative capacity of DM kidneys after acute injury by reversing the hyperglycemic memory 81 5. Discussion 85 6. Future perspectives 92 7. Summary of the work 93 8. References 96 Declaration on the independent preparation of the dissertation 104 Curriculum Vitae 106 List of publications 107 Acknowledgement 109

Diabetic kidney disease (DKD) is the leading cause of end stage renal disease worldwide and occurs in up to 40% of patients with diabetes. The standard of care for DKD treatment has not kept up with the current health epidemic, which has led to a heavy economic toll and substantial health burden. Targeting either cell surface (cs)GRP78, activated α2-macroglobulin (α2M*) or preventing their interaction may provide a novel anti-fibrotic therapeutic target for the treatment of DKD and potentially non-diabetic chronic kidney disease (CKD) as well. Previously our lab has shown that HG-induced csGRP78 is a mediator of PI3k/Akt signaling and downstream extracellular matrix (ECM) protein production in glomerular mesangial cells (MC). However, the ligand responsible for activating high glucose (HG)-induced csGRP78 had not yet been determined. We have shown thus far that α2M is endogenously produced, secreted, and activated (denoted α2M*) in HG by MC, which leads to its binding to and activation thereof csGRP78. Further, α2M knockdown or α2M* neutralization attenuated Akt activation, the production of the profibrotic cytokine connective growth tissue factor (CTGF) and ECM proteins fibronectin and collagen IV. We have also shown that integrin β1 (Intβ1), a transmembrane receptor, associated with csGRP78 under HG conditions and likely acts as a tether to present csGRP78 completely extracellularly on MC. Interestingly, Intβ1 activation, even in the absence of HG, was sufficient to induce csGRP78 translocation. Further, inhibition of either csGRP78 or Intβ1 prevented synthesis, secretion and signaling of TGFβ1. This data implicates a role for Intβ1 as a required signaling partner for csGRP78-mediated profibrotic signaling. To further our understanding of csGRP78/ α2M*’s role in DKD, we investigated their ability to mediate TGFβ1 signaling through its non-proteolytic activator thrombospondin-1 (TSP1). Here, HG-induced TSP1 expression, ECM deposition, and activation of TGFβ1 was regulated by the PI3k/Akt pathway via csGRP78/α2M* in MC. Furthermore, we assessed whether this csGRP78/ α2M* axis is relevant to promoting profibrotic signaling in other renal cell types, including proximal tubule epithelial cells (PTEC) and renal fibroblasts (RF), that contribute to the pathogenesis of both later stage DKD and non-diabetic CKD. We show evidence here that HG and direct treatment with TGFβ1, a key pathologic regulator of kidney fibrosis, induce GRP78 surface translocation as well as the endogenous production and activation of α2M in both PTEC and RF. Inhibition of either csGRP78 or α2M* prevented TGFβ1 signaling measured as Smad3 activation as well as downstream ECM production. Interestingly, inhibition of this pathway under direct TGFβ1 treatment did not prevent Smad3 activation, implicating a role for Smad-independent TGFβ1 signaling through this axis. We identified the known noncanonical TGFβ1 signaling partners, yes associated protein (YAP) and transcriptional co-activator with PDZ binding motif (TAZ), are mediated by csGRP78 and α2M*. Lastly, we evaluated the potential therapeutic benefit of inhibiting csGRP78/α2M* interaction in the kidney fibrosis model, unilateral ureteral obstruction (UUO). Here, we show evidence that inhibition of this signaling axis using an inhibitory peptide can prevent renal fibrosis. Whether this peptide also prevents fibrosis in DKD is currently being assessed. Together, these studies strongly implicate targeting csGRP78/α2M* interaction as a novel anti-fibrotic therapeutic intervention for early and late stage DKD, as well as a potential role in non-diabetic CKD. / Thesis / Doctor of Philosophy (Medical Science) / Diabetic kidney disease is the leading cause of kidney failure in developed nations. This progressive disease leads to the loss of kidney function due to an accumulation of scar proteins in the kidney over time. High glucose is a major factor that causes this to occur. Our lab studies specific kidney cells called mesangial cells, proximal tubule epithelial cells, and fibroblasts that produce scar proteins in the presence of high glucose. We have shown that when these cells are treated with high glucose, this causes the movement of a protein called GRP78 that normally resides inside the cell to move to the cell’s surface where it can interact with other proteins. My research has established that the proteins alpha 2-macroglobulin (ɑ2M), integrin β1 (Intβ1), and thrombospondin-1 (TSP1) can bind to GRP78 on the cell surface and cause cells to make scar proteins. Preventing ɑ2M or Intβ1 from binding to GRP78 or preventing TSP1 production prevents mesangial cells from making scar proteins when exposed to high glucose. In a mouse model that overproduces these scar proteins, we showed that preventing cell surface GRP78 and α2M interaction prevents scar protein production and is thus a novel potential treatment option for kidney disease.

Diabetes mellitus is a metabolic disease largely due to lifestyle and nutritional imbalance, resulting in insulin resistance, hyperglycemia and vascular complications. Diabetic kidney disease (DKD) is a major cause of end-stage renal failure contributing to morbidity and mortality worldwide. Therapeutic options to prevent or reverse DKD progression are limited. Endothelial and glomerular filtration barrier (GFB) dysfunction and sterile inflammation are associated with DKD. Neutrophil extracellular traps (NETs), originally identified as an innate immune mechanism to combat infection, have been implicated in sterile inflammatory responses in non-communicable diseases. However, the contribution of NETs in DKD remains unknown. Here, we show that biomarkers of NETs are increased in diabetic mice and diabetic patients and that these changes correlate with DKD severity. Mechanistically, NETs promote NLRP3 inflammasome activation and glomerular endothelial dysfunction under high glucose stress in vitro and in vivo. Inhibition of NETs (PAD4 inhibitor) ameliorate endothelial dysfunction and renal injury in DKD. Taken together, NET-induced sterile inflammation promotes diabetes-associated endothelial dysfunction, identifying a new pathomechanism contributing to DKD. Inhibition of NETs may be a promising therapeutic strategy in DKD.

Contexte : Les patients âgés atteints de néphropathie diabétique ne reçoivent souvent pas un traitement pharmacologique optimal. Les directives de pratique clinique actuelles n'intègrent pas le concept de soins personnalisés. Les algorithmes d'aide à la décision clinique (ADC) qui tiennent compte à la fois des preuves et des soins personnalisés pour améliorer les résultats des patients peuvent améliorer les soins aux personnes âgées. L'objectif de cette recherche est de concevoir et de valider un algorithme ADC pour la prescription d'inhibiteurs du système rénineangiotensine- aldostérone (ISRAA) pour les patients âgés atteints de diabète. Méthodes : La conception de l'algorithme ADC comprenait trois phases principales. Dans la première phase, nous avons recherché, examiné et évalué les preuves actuelles sur plusieurs sujets liés aux décisions de prescription pour les patients âgés et à l'adhésion des cliniciens aux directives de pratique. Nous avons également procédé à un examen systématique et à une méta-analyse d'essais cliniques randomisés afin de déterminer les valeurs du nombre de patients à traiter (NPT) et du délai d'obtention d'un avantage (DOA) applicables à notre population cible en vue de leur utilisation dans l'algorithme. Dans la deuxième phase, nous avons exploré les points de vue des patients et des prestataires de soins de santé sur les outils ADC en menant des entretiens avec les patients et une enquête transversale auprès des prestataires de soins de santé. Dans la troisième et dernière phase, les résultats des études réalisées dans les phases un et deux ont été utilisés pour informer le développement de l'algorithme ADC qui a ensuite été validé dans une étude Delphi. Résultats : Nous avons créé un algorithme ADC qui couvrait 16 scénarios possibles. Neuf scénarios correspondaient à des recommandations de méta-analyses, tandis que cinq scénarios correspondaient à des directives de pratique clinique. Pour les neuf cas, nous avons généré 36 recommandations personnalisées et neuf recommandations générales sur la base des valeurs NPT et DOA calculées et appariées. En outre, nous avons pris en compte l'espérance de vie et la capacité fonctionnelle du patient. L'algorithme a été validé lors de trois tours d'une étude Delphi. Conclusion : Nous avons conçu un algorithme de CDS fondé sur des preuves qui intègre des considérations souvent négligées dans les directives de pratique clinique, notamment l'espérance de vie restante, la charge médicamenteuse et l'état fonctionnel. Les prochaines étapes consistent à le tester dans le cadre d'un essai clinique afin d'étudier s'il est capable d'atteindre des objectifs cliniques prévisibles et réalistes, de maintenir la qualité de vie des personnes âgées et de réduire l'utilisation et le coût du système de santé. / Background: Older patients with diabetic nephropathy often do not receive optimal pharmacological treatment. Current clinical practice guidelines do not incorporate the concept of personalized care. Clinical decision support (CDS) algorithms that consider both evidence and personalized care to improve patient outcomes can improve the care of older adults. The aim of this research is to design and validate a CDS algorithm for prescribing renin-angiotensin aldosterone system inhibitors (RAASi) for older patients with diabetes. Methods: The design of the CDS algorithm included three main phases. In phase one, we searched, reviewed, and evaluated current evidence on several topics related to prescribing decisions for older patients and clinicians' adherence to practice guidelines. We also conducted a systematic review and a meta-analysis of randomized clinical trials to determine the number needed to treat (NNT) and time-to-benefit (TTB) values applicable to our target population for use in the algorithm. In phase two, we explored the views of patients and healthcare providers on CDS tools through conducting patient interviews and a cross-sectional survey of healthcare providers. In the third and final phase, findings from studies completed in phases one and two were used to inform the development of the CDS algorithm which was then validated using modified Delphi methodology. Results: We have created a CDS algorithm that covered 16 possible scenarios. There were nine scenarios matched to meta-analysis recommendations, while five scenarios were matched to clinical practice guidelines. For the nine cases, we have generated 36 personalized and nine general recommendations based on the calculated and matched NNT and TTB values. In addition, we have considered the patient’s life expectancy and functional capacity. The algorithm was validated in three rounds of a modified Delphi study. Conclusion: We designed an evidenceinformed CDS algorithm that integrates considerations often overlooked in clinical practice guidelines, including remaining life expectancy, medication burden, and functional status. The next steps include testing in a clinical trial to study if it is able to achieve predictable and realistic clinical goals, maintaining quality of life in older adults, and reducing healthcare system use and cost.

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