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Chronic kidney disease is an urgent public health problem in the U.S., affecting 15% of all adults, and more than 740,000 have progressed to end-stage renal disease (ESRD), requiring life-sustaining renal replacement therapy (RRT). ESRD has devastating health, quality-of-life, and economic consequences, rendering most patients unable to maintain employment and costing Medicare $36 billion in 2017. Arguably, the most disadvantaged subgroup is the subset of patients that received no or minimal pre-ESRD nephrology care, which currently accounts for one third of the total ESRD population. This subgroup suffers increased morbidity and mortality, and has limited access to kidney transplantation, the optimal RRT. Despite this subgroup representing a large minority of the ESRD patient population, there has been no U.S.-based examination of their ESRD diagnosis experiences, coping styles and strategies, and RRT decision-making preferences. Therefore, we conducted a study that compared the ESRD diagnosis experiences, coping styles and strategies, and RRT decision-making preferences among patients with varying amounts of pre-ESRD nephrology care. We also assessed nephrologists’ current practices and perspectives on the manner and timing of RRT education for patients with varying amounts of pre-ESRD care. This mixed methods study provides a comprehensive understanding of the diagnosis experiences, coping styles and strategies, and RRT decision-making preferences of patients facing sudden and unexpected ESRD diagnosis. The study contributes important knowledge about this subgroup of patients that can influence and improve health care delivery. The results of this research will inform future intervention-based investigations to improve care for patients with minimal or no pre-ESRD nephrology care. / Public Health

<p> Physical activity improves physical and psychological functioning in patients with chronic kidney disease (CKD). However, no studies have investigated the determinants of physical activity in the CKD population. The purpose of the study was to evaluate the utility of the Theory of Planned Behaviour (TPB) for understanding physical activity in the CKD population. A secondary purpose of this study was to examine alternate conceptualizations of the subjective norm construct within the TPB framework. We hypothesized that attitude, subjective norm (injunctive and descriptive norms), perceived behavioural control (PBC), and social support would predict intention to engage in physical activity and that both intention and PBC would predict physical activity behaviour.</p> <p> Participants (52 male, 28 female, mean age = 68.43 (13.21)) were recruited from nephrologists' clinics and were all predialysis (mean serum creatinine = 310.55 (148.75) μmol/L). Participants completed a questionnaire assessing attitude, subjective norm, PBC, and social support. One week later, participants were phoned for a follow-up interview to assess their physical activity during the preceding week.</p> <p> In a regression model, 61% of the variance in intention to perform physical activity was explained, with PBC (β=.69,p<.001) emerging as the sole significant predictor, while attitude (β=.17, p=.10), subjective norm (β=.02, p=.89), informational support from family (β=-.10,p=.33), and informational support from doctors (β=-.05, p=.54) were non-significant predictors. In a regression model to explain physical activity, 28% of the variance in physical activity was explained, with intention emerging as a significant predictor (β=.53, p=.02), but not PBC (β=.18, p=.29).</p> <p> The hypotheses were only partially supported, as PBC emerged as a significant predictor of physical activity intention, while attitude, subjective norm, and social support did not. Furthermore, intention, but not PBC, predicted physical activity behaviour. These results demonstrate the utility of the TPB for explaining physical activity in the CKD population. Additional research is required to clarify if targeting PBC may be an effective means for intervention to increase physical activity in the CKD population.</p> / Thesis / Master of Science (MSc)

The global prevalence of chronic kidney disease (CKD) has risen at an accelerating rate, increasing the global healthcare burden for long-term and chronic care costs. Multiple risk factors including hypertension, diabetes, and dyslipidemia synergistically induce the progression of CKD. Chief among these factors are dyslipidemia and obesity; increased free fatty acid uptake due to excess consumption of lipid-rich diets has been shown to promote intra-renal lipid accumulation in several in vivo models and in patients in various stages of CKD. Furthermore, patients with renal disease are also at a substantially higher risk for atherosclerotic cardiovascular disease (CVD). In the general population, as well as in patients with renal disease, circulating low-density lipoprotein cholesterol (LDLc) is a well-established driver of atherosclerotic lesion development and CVD progression. In 2003, the proprotein convertase subtilisin/kexin type-9 (PCSK9) was identified as the third locus of familial hypercholesterolemia and was further characterized for its ability to enhance the degradation of the low-density lipoprotein receptor (LDLR). Since this seminal discovery, the development of monoclonal antibodies targeted against PCSK9 demonstrated a significant reduction in LDLc and subsequent CVD risk, establishing the remarkable ‘bench to bedside’ transition. However, the inherent role of PCSK9 in regulating lipid homeostasis remained unknown in different pathological conditions. In the first chapter of my thesis, I demonstrate that PCSK9 regulates the LDLR as a feedback mechanism to protect against non-alcoholic steatohepatitis (NASH) progression induced by a high-fat diet (HFD) challenge. Since its seminal discovery, PCSK9 was also characterized to modulate a wide variety of receptors known to play a crucial role in lipid metabolism including the cluster of differentiation 36 (CD36), the very low-density lipoprotein receptor (VLDLR), and the apolipoprotein E receptor 2 (ApoER2). Previously, we have demonstrated that the absence of PCSK9 promotes diet-induced non-alcoholic steatohepatitis and liver injury through increased surface expression of CD36. Given that these same receptors are well-expressed on renal epithelia, the second chapter of my thesis demonstrates that PCSK9 is also able to modulate renal lipid metabolism by attenuating tubular lipid accumulation and subsequent renal injury. Furthermore, when PCSK9 was first characterized by Seidah and colleagues in 2003, in situ hybridization of murine PCSK9 demonstrated that it was primarily expressed in the liver, but also well-expressed in the kidney cortex, cerebellum, and small intestines. Despite its expression in a wide range of tissues, the secretion of PCSK9 was exclusive to the liver, thus, questioning what the intracellular role of PCSK9 may be. Hence, my last chapter of my masters studies lies in establishing the role of intracellular PCSK9 expression in a cellular process known as endoplasmic reticulum (ER) stress in the kidney. ER stress is a phenomena which primarily occurs due to increased accumulation of misfolded polypeptides, and has been implicated in numerous metabolic diseases including hepatic steatosis, CKD, and neurodegenerative pathologies. Previously, we have demonstrated that overexpressing wild-type and variants of PCSK9 in a Pcsk9-/- mouse does not induce the activation of the unfolded protein response (UPR) and attenuates hepatic ER stress. Using a well-established CKD model, I show that Pcsk9-/- mice exhibit increased renal ER stress and injury relative to wild-type controls. Overall, my findings demonstrate for the first time that both extracellular and intracellular PCSK9 has the ability to modulate renal injury using two distinct mechanism to protect against CKD progression. / Thesis / Master of Health Sciences (MSc)

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

Kidney fibrosis is a major culprit in the development and progression of chronic kidney disease (CKD), ultimately leading to the irreversible loss of organ function. Thymocyte differentiation antigen-1 (Thy-1) controls many core functions of fibroblasts relevant to fibrogenesis but is also found in a soluble form (sThy-1) in serum and urine. We investigated the association of sThy-1 with clinical parameters in patients with CKD receiving hemodialysis treatment compared to individuals with a preserved renal function. Furthermore, Thy-1 tissue expression was detected in a mouse model of diabetic CKD (eNOS-/-; db/db) and non-diabetic control mice (eNOS-/-). Serum and urinary sThy-1 concentrations significantly increased with deteriorating renal function, independent of the presence of diabetes. Serum creatinine is the major, independent, and inverse predictor of serum sThy-1 levels. Moreover, sThy-1 is not only predicted by markers of renal function but is also itself an independent and strong predictor of markers of renal function, i.e., serum creatinine. Mice with severe diabetic CKD show increased Thy-1 mRNA and protein expression in the kidney compared to control animals, as well as elevated urinary sThy-1 levels. Pro-fibrotic mediators, such as interleukin (IL)-4, IL-13, IL-6 and transforming growth factor β, increase Thy-1 gene expression and release of sThy-1 from fibroblasts. Our data underline the role of Thy-1 in the control of kidney fibrosis in CKD and raise the opportunity that Thy-1 may function as a renal antifibrotic factor.

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

Faible masse musculaire a été peu étudiée chez les patients avant le stade terminal de la maladie rénale chronique (MRC). Nous avons évalué la masse musculaire à partir de la créatininurie des 24h pour étudier ses déterminants, son évolution avec le déclin de la fonction rénale ainsi que ses liens avec les risques de progression vers l’insuffisance rénale terminale traitée (IRTT) et de décès avant IRTT. Dans la cohorte NephroTest incluant 1429 patients avec une MRC stades 1 à 4, le débit de filtration glomérulaire a été mesuré par la clairance du 51Cr-EDTA (DFGm) et estimé par l’équation CKD EPI (DFGe). La créatininurie moyenne à l’inclusion diminuait de 15.3±3.1 à 12.1±3.3 mmol/24 chez les hommes et de 9.6±1.9 à 7.6±2.5 chez les femmes, pour une baisse du DFGm de ≥ 60 à < 15 mL/min/1.73 m2. Être plus âgé, avoir un diabète, un faible IMC ou un niveau faible de protéinurie et d’apports protidiques était associé à un niveau faible de créatininurie. Un déclin annuel du DFGm de 5 mL/min/1.73 m2 était lié à une baisse de créatininurie, indépendamment de ces déterminants. Au cours d’un suivi médian de 3.6 ans, 229 patients ont développé une IRTT, et 113 sont décédés avant IRTT. Après ajustement sur les facteurs de confusion, le hasard ratio (HR) était de 1.6 (0.88-2.9) pour le risque de décès et de 0.60 (0.39-0.91) pour le risque d’IRTT, dans le 1er vs 4ème quartile de créatininurie. La baisse de la créatininurie apparait précocement dans la MRC et est liée au décès avant dialyse. La diminution du risque d’IRTT pourrait s’expliquer par un démarrage plus tardif de la dialyse en raison d’une surestimation du DFGm par le DFGe chez les patients avec une faible créatininurie. / Mainly described in patients on dialysis, muscle wasting has received little attention in early stage chronic kidney disease (CKD). We used 24-hour creatininuria to assess determinants of low muscle mass and its putative associations with CKD outcomes, using data from the NephroTest cohort, including 1429 non-dialysis patients with CKD stages 1 to 5. Kidney function was assessed with both measured (mGFR, by 51Cr-EDTA renal clearance) and estimated glomerular filtration rate (eGFR, by CKD-EPI equation). End-stage renal disease (ESRD) and pre-ESRD death were the main studied outcomes. The mean baseline creatininuria decreased from 15.3±3.1 to 12.1±3.3 mmol/24 h in men and from 9.6±1.9 to 7.6±2.5 in women, when mGFR fell from ≥ 60 to < 15 mL/min/1.73 m2. Other determinants of low creatininuria were an older age, diabetes, a lower body mass index, a lower level of proteinuria or protein intake. A fast annual decline in mGFR of 5 mL/min/1.73 m2 was linked with a 2-fold decrease in creatininuria, independent of changes in protein intake and other determinants of muscle mass. Over a median follow-up of 3.6 years, 229 patients developed ESRD and 113 patients died before ESRD. After adjustment for confounders, patients with low muscle mass showed a significantly higher risk for pre-ESRD death (HR 1.6, 95% CI 0.88-2.9), but a lower risk for ESRD (HR 0.60, 95% CI 0.39-0.91). The latter was reversed (HR 1.5, 95% CI 1.01-2.4) when mGFR was replaced by eGFR. Decrease in 24-hour creatininuria may appear early in CKD patients, is related to pre-ESRD death. The lower risk for ESRD may reflect later dialysis start due to overestimation of true GFR by eGFR in patients with low muscle mass.

Autosomal dominant polycystic disease (ADPKD) is the most common hereditary renal disease and is characterized by renal cyst growth and enlargement. Hypertension occurs early when renal function is normal and is characterized by decreased renal blood flow. Accordingly, the measurement of blood flow in the renal arteries can be a valuable tool in evaluating disease progression. In studies performed in conjunction with this work, blood flow was measured through the renal arteries using magnetic resonance imaging (MRI). In order to validate these in vivo measurements, a vascular phantom was created using polyvinyl alcohol (PVA) and also scanned using MRI under controlled steady flow conditions. Ranges of vessel diameters and flow velocities were used to simulate actual flow in a normal and diseased population of adults and children. With the vessel diameters studied in this experiment, minimization of field of view and an increase in spatial resolution is important in obtaining accurate data. However, a significant difference does not exist between the results when using the 160 or 200 mm FOV. An increase in the number of phase encodings provides improved results, although an increase in image acquisition time is observed. Velocity-encoding in all three orthogonal directions does not improve image data. This method of using MRI to measure flow through a vessel is shown to be both accurate and reproducible, and the protocol providing the most correct results is prescribed. Breath-hold phase-velocity encoded MRI proves to be an accurate and reproducible technique in capturing flow and has the potential to be used for the purpose of observing hemodynamic changes in the renal arteries with the progression of ADPKD.