京都大学 / 新制・課程博士 / 博士(医学) / 甲第24795号 / 医博第4987号 / 新制||医||1066(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 小林 恭, 教授 波多野 悦朗, 教授 羽賀 博典 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(社会健康医学) / 甲第24536号 / 社医博第128号 / 新制||社医||12(附属図書館) / 京都大学大学院医学研究科社会健康医学系専攻 / (主査)教授 近藤 尚己, 教授 西浦 博, 教授 柳田 素子 / 学位規則第4条第1項該当 / Doctor of Public Health / Kyoto University / DFAM

Chronic kidney disease (CKD) is a worldwide health burden with increases risk of end-stage renal function if left untreated. CKD induced in the context of metabolic syndrome (MS) increases risks of hypertension, hyperglycemia, excess body fat and dyslipidemia. Our Centre previously generated a renin-dependent hypertensive/ type 1 diabetic mouse model and lead to the development of several signs associated with human diabetic kidney disease (DKD), however the extent and impact of dyslipidemia in this model remains unknown. We hypothesized that combining a high-fat diet (HFD) regimen onto the hypertensive/ diabetic phenotype would mimic features of MS induced-CKD in mice. An 8-week-old male genetically hypertensive mice (Lin+) were subjected to streptozotocin (STZ) intraperitoneal (i.p.) injections (50 mg/kg, 5 days consecutive) to induce hyperglycemia. Four-weeks later hypertensive/ diabetic mice (Lin+ mouse with induced beta cells death, also known as LinSTZ) were fed a 60% kCal HFD for 8 weeks. This study shows that HFD-fed LinSTZ mice developed less glomerular hypertrophy, scarring and albuminuria and hepatocytes fat accumulation at endpoint than regular-diet fed littermates. Moreover, antidiabetic drug Canagliflozin, dosed at 30 mg/kg body weight, showed reno-protection in the LinSTZ mice model. Taken together, our results show that LinSTZ mice fed a HFD did not lead to a more robust model of MS induced CKD. In fact, several indices of renal injury were reduced by feeding LinSTZ mice a HFD or treating them with Canagliflozin.

The study of ferritin includes a rich history of discoveries and scientific progress. Initially, the composition of ferritin was determined. Soon, it was shown that ferritin is a spherical, hollow protein. Eventually, over several decades of research, the structure and some function of this interesting protein was elucidated. However, the ferritin field was not completely satisfied. Today, for example, researchers are interested in refining the details of ferritin function, in discovering the role of ferritin in a variety of diseases, and in using ferritin for materials chemistry applications. The work presented in this dissertation highlights the progress that we have made in each of these three areas: 1) Mechanistic studies: The buffer used during horse spleen ferritin iron loading significantly influences the mineralization process and the quantity of iron deposited in ferritin. The ferrihydrite core of ferritin is crystalline and ordered when iron is loaded into ferritin in the presence of imidazole buffer. On the other hand, when iron is loaded into ferritin in the presence of MOPS buffer, the ferrihydrite core is less crystalline and less ordered, and a smaller amount of total iron is loaded in ferritin. We also show that iron can be released from the ferritin core in a non-reductive manner. The rate of Fe3+ release from horse spleen ferritin was measured using the Fe3+-specific chelator desferoxamine. We show that iron release occurs by three kinetic events. 2) Disease studies: In order to better understand iron disruption during disease states, we performed in vitro assays that mimicked chronic kidney disease. We tested the hypothesis that elevated levels of serum phosphate interrupted normal iron binding by transferrin and ferritin. Results show that phosphate competes for iron, forming an iron(III)-phosphate complex that is inaccessible to either transferrin or ferritin. Ferritin samples separated from the iron(III)-phosphate complex shows that as the phosphate concentration increases, iron loading into ferritin decreases. 3) Materials chemistry studies: Anion sequestration during ferritin core reduction was studied. When the core of horse spleen ferritin is fully reduced using formamidine sulfinic acid, a variety of anions, including halides and oxoanions, cross the protein shell and enter the ferritin interior. Efforts have been made to use ferritin to control the concentration of anions for reactions. In addition, the native ferrihydrite mineral core of ferritin is a semi-conductor capable of catalyzing oxidation/reduction reactions. Light can photo-reduce AuCl4- to form gold nanoparticles (AuNPs) with ferritin as a photocatalyst. The mechanism of AuNP formation using ferritin as a photocatalyst was examined. From this work, we propose that the ferrihydrite core of ferritin photo-reduces; the mineral core dissolves into a soluble iron(II) mineral. The iron(II) then re-oxidizes, and a new mineral forms that appears to be the new photocatalyst, as the lag phase is significantly decreased with this new mineral form of ferritin.

No description available.

This report seeks to describe the status of kidney disease and renal replacement therapy in lower-resource settings, particularly sub-Saharan Africa. Acute kidney injury and transplantation are included on a limited basis because it is impossible consider the renal replacement therapy landscape at the exclusion of either. As in the rest of the developing world, chronic kidney disease and end-stage renal disease place a sizable and rapidly growing burden on sub-Saharan Africa, and Africans face a double-burden of disease from communicable and non-communicable diseases. Meanwhile, renal replacement therapy and the subspecialty of nephrology are expanding in sub-Saharan Africa, from non-existence in many countries to a limited, tentative subsistence, largely with the support of international organizations and the dedication of local nephrologists. Hemodialysis is the most common form of renal replacement therapy in sub-Saharan Africa, but peritoneal dialysis services, particularly for acute kidney injury, are growing and renal transplants are performed in a few sub-Saharan countries. Nonetheless, in the majority of sub-Saharan Africa, maintenance dialysis is still only available to the wealthy urban few. Although peritoneal dialysis may seem more feasible in the developing world than hemodialysis for multiple reasons, it is still fraught with challenges that make widespread implementation presently unadvisable. As renal replacement therapy is costly and currently unaffordable on a large scale for most of these countries, emphasis must be on identifying at-risk populations through screening and low-cost treatment or management of risk factors to mitigate chronic kidney disease.

While cognitive impairment is common in peripheral diseases such as chronic kidney disease (CKD), mechanistic insights and effective therapies are lacking. Multiple toxins accumulating as a consequence of CKD have been identified, yet the consequences for cellular crosstalk in the brain and the mechanisms underlying the associated neuronal dysfunction remain largely elusive. In the case of CKD, more than 100 uremic toxins have been identified. Renal transplantation largely reverses the cognitive impairment associated with CKD, demonstrating that cognitive impairment in CKD can be reversed. This indicates that pharmaceutical approaches to target cognitive impairment in association with CKD may be feasible. However, it is unlikely that targeting a single toxin will be sufficient to combat neuronal dysfunction associated with peripheral diseases such as CKD, given the large number of toxins involved and since the pattern of accumulating toxins varies among affected patients. Rather than identifying single toxins, identifying a common mechanism inducing neuronal dysfunction and thus impairing cognition may identify new and feasible therapeutic approaches. One commonality of peripheral diseases such as liver or renal failure is sterile inflammation. Sterile inflammation has been linked with neurodegenerative diseases and associated cognitive impairment and inflammasome activation is one hallmark of chronic pathologies in the brain. Mutations in the inflammasome component NLRP3 show clinical manifestations of cryopyrin- associated periodic syndromes (CAPS), which are characterized by skin rash, fever and joint pain. Further, abnormal and constant NLRP3 signaling has been associated with some chronic and degenerative diseases such as Alzheimer’s disease (AD), atherosclerosis, arthritis or cancer. A causative function of the NLRP3 inflammasome for neurodegenerative processes is supported by preclinical studies. These pre-clinical studies used whole body knock out mice to demonstrate that deficiencies of NLRP3, caspase-1 or the primary receptor for IL-1β, IL-1R1, protect mice from neurodegenerative processes. While providing important insights into the role of the NLRP3-inflammasome in neurodegenerative processes, these studies did not identify the relevant cell types in which the inflammasome is activated, the mechanisms underlying inflammasome activation and the consequences thereof, e.g. for intracerebral cross-talk. In addition, whether sterile inflammation triggered by the NLRP3 inflammasome impairs cognition in the setting of primarily peripheral diseases such as CKD remains unknown. To address these open questions, I used a mouse model of CKD, in which I detected NLRP3 inflammasome in brains. Interestingly, despite inflammasome activation in the brain, microglial caspase-1 deficiency did not improve cerebral inflammation and cognition in CKD mice. I identified noncanonical IL-1β maturation in microglia in CKD conditions, which was cathepsin c – caspase-8 mediated. Restoring K+ homeostasis in microglia or genetic inhibition of neuronal IL-1R1 signaling abolished CKD-induced cognitive impairment. Mechanistically, noncanonical IL-1β maturation and secretion from microglia promotes via IL-1R signaling cognitive impairment in neurons. This identifies a molecular mechanism of sterile CNS inflammation and the associated intercellular signaling pathway, which may be therapeutically amendable. Microglial K+ dyshomeostasis and noncanonical microglial IL-1β maturation may be druggable targets in some forms of cognitive impairment.:Content 2 List of abbreviations 5 Graphical abstract 8 2 Introduction 9 2.1 Chronic kidney disease and cognition 11 2.2 Microglia cells 13 2.3 The inflammasome, potassium dyshomeostasis in brain cells and thallium autometallography 15 2.4 Sterile inflammation in neurodegenerative diseases 17 3 Aims of the study 19 4 Materials and Methods 20 4.1 Reagents 20 4.2 Mice 27 4.3 CKD mouse model (5/6 nephrectomy model) 30 4.4 Evans Blue extravasation assay 32 4.5 2-photon microscopy 32 4.6 Analysis of mice 33 4.7 In vivo interventions 33 4.8 Histology and immunohistochemical analysis 34 4.9 Cell culture 34 4.10 Dextran permeability assay 35 4.11 Thallium-AMG (TlAMG), ex vivo and in vitro 36 4.12 Protein extraction and Western blotting 38 4.13 IL-1β ELISA 38 4.14 Reverse Transcriptase Polymerase Chain Reaction (RT–PCR) 38 4.15 Proximity ligation assay (PLA) 39 4.16 Behavioral analysis 39 4.17 Cathepsin c substrate assay 40 4.18 snRNA-Seq 41 4.19 Statistical Analysis 42 5 Results 43 5.1 Chronically impaired renal function leads to cognitive decline 43 5.2 Blood brain barrier (BBB) disruption in chronic kidney disease 44 5.3 Potassium dyshomeostasis in brain cells in CKD 45 5.4 CKD leads to microglia activation 46 5.5 Priming of microglia in CKD depends on potassium dyshomeostasis and its restoration improves cognition in CKD 49 5.6 TRAM34 ameliorates potassium dyshomeostasis and behavior in CKD 51 5.7 Uremia-induced cognitive impairment depends on microglia- neuron crosstalk via IL-1R1 52 5.8 Deciphering the microglial molecular pathway in CKD 56 5.9 Microglia activation in CKD is independent of NLRP3 56 5.10 Microglial IL-1β maturation occurs independently of the NLRP3-Caspase-1 inflammasome in CKD 57 5.11 The role of caspase- 8 in microglia activation in CKD 60 5.12 Lysosomal cathepsin c promotes microglia activation pivotal for caspase-8 activation 62 5.13 Broader implication of the pathway in other chronic peripheric diseases 63 5.14 Microglia inflammasome activation and IL-1β release is sufficient to induce cognitive impairment 64 5.15 Tables 66 6 Discussion 69 7 Summary 75 8 Zusammenfassung 80 9 References 86 10 Declaration about the independent preparation of the work 97 11 Presentation of own contribution 98 12 Curriculum vitae 99 13 Publications 104 14 Acknowledgments 106

Chronic kidney disease (CKD) is a complex and progressive condition with limited curative therapies and is associated with both physical comorbidity, impaired health-related quality of life, and financial strain on the healthcare system. Currently, CKD is defined by a fixed threshold of an estimated glomerular filtration rate (eGFR) < 60 mL/min/1.73m2, which coincides with approximately 60% of healthy kidney function, for ≥3 months regardless of age. However, this definition does not account for natural declines in kidney function with advanced age, leaving older individuals (ages >65 years) with naturally lower eGFR and without significant kidney damage being over-diagnosed with CKD. Conversely, there is also concern of underdiagnosis of CKD in younger adults (ages <40 years) with “modest” eGFR reductions (eGFR levels well above 60, but below age-expected values). Indeed, severe impairment is not detected in younger adults until they lose close to 50% of their healthy kidney function, precluding timely prevention of CKD progression and its associated complications (premature mortality, cardiovascular events, etc.). However, whether these “modest” eGFR reductions are associated with elevated clinical risk in younger adults is unknown. This thesis is based on a retrospective cohort study using linked healthcare administrative databases to examine the association of index eGFR categories with time to adverse outcomes, relative to age-specific referents. In the first manuscript, we compared associations with key adverse outcomes (all-cause mortality, cardiovascular events, and kidney failure) and patterns of healthcare utilization between younger (ages 18-39), middle-aged (40-49), and older adults (50-65 years). In the second manuscript, we examined associations with major cardiovascular events (cardiovascular mortality, acute coronary syndrome, ischemic stroke, heart failure) by age group. In both manuscripts, we noted significant elevations in risk of adverse outcomes at higher eGFR levels relative to age-specific referents in younger, compared to middle-aged and older adults. Despite this age-related vii disparity in clinical risk with modestly reduced eGFR, younger adults were least likely to obtain a repeated eGFR measure or be referred to a specialist during follow-up. Notably, these findings persisted for individual adverse events and in clinically important subgroups, as well as after various sensitivity analyses (adjusting for additional comorbidities, defining index eGFR using repeated measures, using common referents, and excluding individuals with different underlying mechanisms for reduced eGFR (pregnancy, acute kidney injury, etc.)). The current thesis presents evidence of elevated clinical risk with modest reductions in kidney function in younger adults, emphasizing the importance of risk-based eGFR thresholds that vary with age and considering modestly reduced eGFR as important cardiovascular risk factors worth monitoring in routine clinical practice.

This systematic review aims to examine the relationship between the social determinants of health that significantly impact the prevalence and progression of chronic kidney disease (CKD) amongst Black Americans. The Black American population has the highest prevalence of CKD in the United States, while concurrently possessing prominent genetic risk factors for this chronic disease. The social determinants: healthcare quality and access (extended to account for health behaviors), social and community context, and economic stability were specifically assessed in this review. Key terms were utilized to search electronic databases PubMed and Web of Science, which yielded 470 unduplicated articles. Twenty-nine articles met the inclusion criteria for this systematic review, with three articles being applicable to the three selected social determinants, six being applicable to social and community context, six being applicable to economic stability, and fourteen being applicable to healthcare quality and access. Major contributors towards CKD incidence and progression amongst Black Americans were identified to be: exposure to discrimination, expectations of discrimination and prejudice, low routine medical care, limited health literacy, distrust of health providers, being of low socioeconomic status, and a lack of engagement in functional health behaviors (fruit/vegetable consumption and CKD screening). Each social determinant was seldom observed to be operating exclusively as a contributing factor towards CKD, exemplifying how the intersectionality of these factors contributes to increased CKD risk and progression. Findings from this systematic review highlight the need for targeted healthcare initiatives for Black Americans to remedy the CKD endemic.

Chronic kidney disease (CKD) is growing global public health problem affecting 1 in 10 adults in developed countries and recognised as an important risk factor for cardiovascular disease (CVD) development. CVD is the main cause of death among CKD patients. Endothelial injury and dysfunction are critical steps in atherosclerosis, a major CVD. Oxidative stress (increased level of reactive oxygen species, ROS) has been associated with CVD development. Intravenous (IV) iron preparations are widely used in the management of CKD mediated anaemia, and have been associated with increased oxidative stress and cellular dysfunction. This study examined the effect of pharmacologically-relevant concentrations of IV Venofer (iron sucrose) or IV Ferinject (Ferric carboxymaltose, FCM) on primary human umbilical vein endothelial cell (HUVEC) activation/damage and on intracellular ROS generation as well as studying the potential mechanisms responsible. Data from TUNEL assay and Annexin V-FITC/PI staining showed that, IV FCM had no effect, but IV iron sucrose increased HUVEC apoptosis at 24hr. IV iron sucrose inhibited cell proliferation and reduced cell viability. Both compounds induced EC activation through sustained activation of p38 MAPK and up-regulation of ICAM-1 and VCAM-1. Additionally, the compounds induced significant increase in total ROS and superoxide anion production, which was attenuated by the anti-oxidant N-acetylcysteine (NAC). P38 MAPK showed up-regulation of pro-apoptotic protein Bax and down-regulation of antiapoptotic Bcl-2 protein in HUVEC treated with IV iron sucrose and p38 inhibition reversed these effects. In summary, these results suggest that IV iron sucrose causes more severe EC injury than IV FCM. However, both IV iron preparations induced intracellular ROS and superoxide anion generation in HUVEC leading to EC activation/dysfunction, providing a potential explanation for vascular damage in CKD patients.