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Diabetic nephropathy represents the leading cause of end stage renal disease worldwide and requires a kidney transplant or dialysis to survive. The number of patients suffering from diabetes is expected to increase, thus the number of patients with diabetic nephropathy is expected to concomitantly increase. Current treatment for diabetic nephropathy is not sufficient to prevent disease progression in most patients thus there is a need to develop novel therapies to treat diabetic nephropathy. The earliest changes that occur during the pathogenesis of diabetes occur in the glomerulus. The mesangial cells are a subpopulation of cells in the glomerulus that are responsible for coordinating responses with other nearby cell types. Transforming growth factor (TGF)-β1 is a cytokine that mesangial cells secrete, and has been identified as a profibrotic factor during the pathogenesis of diabetic nephropathy. Concerns have been raised in the use of direct anti-TGF-β1 therapy due to adverse events (such as dyspepsia and diarrhea) and lack of efficacy of anti-TGF-β1 monoclonal antibody LY2382770 in patients with diabetic nephropathy. Thus, therapy aimed at modulating TGF-β1 expression or activity may be efficacious in the treatment of diabetic nephropathy while avoiding potential adverse effects. The hypothesis of this thesis is that SREBP-1 and cell surface GRP78 are novel regulators of TGF-β1 signaling in mesangial cells. Our first study aims to define a novel pathway by which SREBP-1 regulates TGF-β1 signaling in kidney mesangial cells. Our results indicate that SREBP-1 regulates the expression of the type I TGF-β1 receptor through its secretion in exosomes. Our second study expands on these findings and aims to determine if inhibition of SREBP in vivo with the inhibitor fatostatin may prevent diabetic nephropathy. Our results indicate that treatment with fatostatin does not prevent diabetic nephropathy, but accentuates kidney injury in non-diabetic mice. Preliminary results from our lab have indicated that under diabetic conditions, GRP78 is upregulated at the cell surface and may contribute to the activation of SREBP-1 in an ER-stress dependent mechanism. Our third study thus aims to characterize the expression of cell surface GRP78 in diabetic conditions, and to determine its pathological relevance in the development of diabetic nephropathy. Our results have established novel pathways by which TGF-β1 signaling is regulated in mesangial cells. This will assist in identification of novel therapeutic targets that may be of use in the treatment of diabetic nephropathy. / Thesis / Doctor of Philosophy (PhD) / Diabetic kidney disease is the leading cause of end stage renal disease and represents an important risk factor for mortality. The goal of this thesis is to understand and describe the pathways and mechanisms that contribute to the development of diabetic kidney disease in order to identify novel therapeutic targets. This thesis has identified the protein sterol regulatory element binding protein (SREBP)-1 and the cell surface presentation of another protein, the 78 kDa glucose regulated protein (GRP78), as contributors to diabetic kidney disease. Furthermore, this thesis has demonstrated that anti-SREBP therapy with the drug fatostatin did not prevent diabetic kidney disease. These studies show that while inhibiting SREBP-1 and cell surface GRP78 may be effective in the treatment of diabetic kidney disease, the drug fatostatin should not be used for treatment.

Diabetic nephropathy (DN) affects around 40% of diabetic patients worldwide and has become a major health concern due to its high morbidity and mortality. The progression of DN is characterized by the thickening of glomerular basement membrane, albuminuria and the development of glomerulosclerosis. Renal function is eventually compromised. Due to various hemodynamic and metabolic changes, especially the elevated blood glucose level in diabetic patients, glomerular mesangial cells have been shown to upregulate transforming growth factor-β1 (TGF-β1) level and signaling, resulting in the excessive production of extracellular matrix (ECM) proteins. The atypical expression of the 78-kDa glucose-regulated protein (GRP78) on the cell surface may be associated with this pro-fibrotic effect through its interaction with the TGF-β1 activation process. However, there is no current literature demonstrating the role of cell surface GRP78 (csGRP78) in the pathogenesis of diabetic renal diseases. The purpose of my MSc project was to determine the role of csGRP78 in TGF-β1 synthesis and activation and thereby in the progression of DN. We hypothesized that the increased expression of csGRP78 in response to high glucose exposure stimulates TGF-β1 upregulation through intracellular signaling, as well as its activation through interaction with the latent complex, which leads to the expansion of mesangial matrix. / Thesis / Master of Science (MSc) / Diabetic kidney disease affects around 40% of diabetic patients worldwide and is a major health concern. A major feature of the disease is glomerulosclerosis, which is the scarring of glomeruli. The glomeruli filter blood passing through blood vessels in the kidneys to remove waste, which will then be excreted into urine. In diabetic patients, high blood glucose causes the fibrosis of glomeruli and damages the filtration barrier. As a result, a large amount of proteins leak from the blood into the urine. It has been discovered that TGF-β1 is one of the key molecules mediating the generation of scar tissue in the glomerulus. It promotes the growth of mesangial cells, a major type of kidney glomerular cells, and stimulates their production of extracellular matrix proteins. Our results showed that GRP78, a protein that is primarily expressed in the endoplasmic reticulum and assists with protein folding, moves from the inside of cells to the surface in response to a high glucose environment. Here, we found that it facilitated TGF-β1 signaling. Based on our studies, we propose that when GRP78 is at the cell surface, it enables the release of latent TGF-β1, increasing TGF-β1 activity and thus promoting the development of disease.

Kidney transplantation is an effective treatment for renal disease that was previously fatal. However, the demand for donor kidneys far exceeds the supply. Due to the scarcity of volunteer donors, the cadaver organs that are retrieved must be optimally utilized. By expanding organ retrieval and sharing pools and improving donor-patient matching algorithms, the utilization of donated organs is enhanced and encouraging medical results are obtained. However, the benefits of enlarged donor and recipient pools may be offset by increasing complexity and decreasing efficiency in the organ distribution process thus increasing cold ischemia time. It is critical to improve distribution process efficiency in order to minimize the time taken to complete the entire process, and thus further enhance patient and graft survival. I attempt to apply supply chain management concepts, agent technologies, mobile communication technologies and decision-making theory to improve the efficiency of the cadaver kidney distribution process. In this thesis I analyze what are the bottlenecks in current cadaver kidney distribution and investigate how agent technology can be applied to improve this process. I propose a distributed multi-agent system operating in a mobile and wireless communication environment to assist transplant coordinators in coordinating with multi-parties in this time-critical distribution process. A prototype system has been developed to help transplanting coordinators in allocating the kidney recipient. / Thesis / Master of Science (MS)

M.Sc. Thesis Dissertation, August 2019, McMaster University / Renal dysplasia, defined as the abnormal development of kidney tissue, is the leading cause of kidney disease in children. While there are numerous causes of renal dysplasia (i.e. genetic, environmental and epigenetic factors), there is no cure to this abnormal defect. Kidney development occurs by two main processes: branching morphogenesis, which forms the collecting duct system, and nephrogenesis, which generates the nephrons, the functional units of the kidney. Our previous studies have demonstrated that β-catenin, a dual-function protein involved in cell adhesion and gene transcription, regulates branching morphogenesis and nephrogenesis. Furthermore, we discovered that nuclear β-catenin levels are increased in kidneys from patients with renal dysplasia, suggesting β-catenin can be a potential therapeutic target to modulate kidney development and renal dysplasia. Quercetin is a flavonoid that reduces β-catenin levels and inhibits its transcriptional activity, leading to improved outcomes in cancer and in kidney fibrosis. The role of quercetin in kidney development and in abnormal defects that arise during kidney development is yet to be examined. Using embryonic mouse kidney organ culture, I found that quercetin treatment resulted in a dose-dependent disruption in branching morphogenesis and nephrogenesis. In addition, quantitative reverse-transcriptase PCR revealed a decreased expression of β-catenin target genes essential for kidney development (i.e. Pax2, Six2 and GDNF). Immunohistochemistry for β-catenin demonstrated that quercetin reduced nuclear β-catenin expression and increased cytoplasmic and membrane-bound expression in a dose-dependent manner. These results were confirmed by Western blot analysis. These novel findings demonstrate that quercetin treatment resulted in decreased levels of nuclear β-catenin, resulting in a decrease in its transcriptional activity which manifested in alterations in kidney developmental processes, suggesting quercetin is effective at reducing nuclear β-catenin in wild-type embryonic kidneys. Next, to determine whether quercetin has any effects on renal dysplasia, I utilized transgenic mice models that overexpress β-catenin in select cells of the embryonic kidney. These models recapitulate the defects observed in human renal dysplasia, including disorganized branching morphogenesis and disrupted nephrogenesis. Quercetin treatment of embryonic dysplastic kidneys resulted in a partial rescue of renal dysplasia which was evident in marked improvements in branching morphogenesis and nephrogenesis, as well as an increase in the number of properly-developing nephrons in the kidney tissue. Analysis of β-catenin expression in quercetin-treated dysplastic kidneys revealed a decrease in nuclear levels and an increase in cytoplasmic and membrane-bound levels, resulting in a reduced expression of target genes (Pax2, Six2, and GDNF). Finally, this partial rescue of renal dysplasia was associated with an improved and organized E-cadherin expression in quercetin-treated dysplastic kidneys, suggesting a possible molecular mechanism of quercetin action in resolving abnormal kidney development. Overall, my findings demonstrate, for the first time, that quercetin reduces β-catenin transcriptional activity in normal and dysplastic kidneys and reduces the severity of defects in renal dysplasia. / Thesis / Master of Science in Medical Sciences (MSMS)

Chronic kidney disease (CKD) is a major cause of morbidity and mortality, affecting more than 10% of the world’s population. CKD is associated with excessive renal fibrosis, which leads to declining kidney function and eventual kidney failure. In CKD, glomerular mesangial cells (MC), resident fibroblasts and tubular epithelial cells undergo phenotypic activation and transition in response to profibrotic and proinflammatory cytokines such as transforming growth factor β1 (TGFβ1). These activated renal cells excessively produce extracellular matrix (ECM) proteins that replace functional renal tissue and lead to renal fibrosis. Caveolae are small omega-shaped invaginations of the plasma membrane that mediate signaling transduction events. Formation of caveolae require the protein caveolin-1 (cav-1). We have previously shown that the ability of MC to produce matrix proteins is dependent on cav-1 expression. Unfortunately, clinically targeting cav-1 within the kidneys, specifically within MC, is technically challenging and as of yet unfeasible. Thus, to better understand how cav-1 deletion is protective, we carried out a microarray screen comparing cav-1 wild-type (WT) and knockout (KO) MC. Here, we discovered significant up-regulation of a TGFβ superfamily inhibitory protein, follistatin (FST). FST specifically targets and neutralizes activin A (ActA) but not TGFβ1. TGFβ1 and ActA both belong to the TGFβ superfamily of cytokines and growth factors. While TGFβ1 itself is a known key mediator of renal fibrosis, therapies aimed at directly inhibiting TGFβ1 in kidney diseases have not been successful due to opposing profibrotic and anti-inflammatory effects. ActA has been shown to act as a strong profibrotic and proinflammatory agent in various organs, including the lungs and liver. We along with others have observed elevated levels of ActA within the kidneys and serum of mice and humans with CKD. Functionally, ActA has been shown to contribute to ECM production in the kidneys. Hence, we hypothesized that ActA inhibition through FST could prove beneficial in CKD. In this thesis, our first study elucidated a novel molecular pathway by which cav-1 regulates expression of the FST in MC. Our results indicate that FST is negatively regulated by cav-1 through a PI3K/PKC zeta/Sp1 transcriptional pathway. Our second study expands on these findings and tests whether exogenous FST administration protects against the progression of CKD in a surgical mouse model of CKD. Here, we discovered that FST acts as a reactive oxygen species (ROS) scavenger and that exogenous administration of FST protects against the development of CKD through the inhibition of renal fibrosis and oxidative stress. Lastly, our third study determined whether microRNAs (miRNAs) are implicated in post-transcriptionally regulating FST through cav-1 and whether these FST-targeting miRNAs can be utilized therapeutically to protect against the development and progression of CKD. Here, we determined that a FST-targeting miRNA, microRNA299a-5p, is significantly downregulated in cav-1 deficient MC, upregulated in vivo in a mouse model of CKD and that its inhibition, in vitro and in vivo protects against the accumulation of ECM proteins and renal fibrosis. These studies collectively suggest that FST is an effective therapeutic option for the management of CKD. / Thesis / Doctor of Philosophy (PhD) / Chronic kidney disease results from excessive fibrosis (scarring) within the kidneys. The goal of this thesis is to understand the molecular mechanisms involving the regulation of an antifibrotic protein, follistatin, in glomerular mesangial cells and to identify its therapeutic potential in chronic kidney disease. This thesis has identified that follistatin, an endogenous inhibitor of the profibrotic cytokine activin A, is regulated transcriptionally by Sp1 and post-transcriptionally by microRNA299a-5p. Furthermore, this thesis has demonstrated that exogenous recombinant follistatin administration protects against the progression of chronic kidney disease and that microRNA299a-5p targeting may be an alternative approach to block renal fibrosis. These studies collectively show that follistatin is an effective treatment for the management of chronic kidney disease.

Renibacterium salmoninarum, the etiological agent of bacterial kidney disease (BKD) of salmonid fish, is a pathogen of great concern among fisheries and the aquaculture industry worldwide. Previous investigations have indicated the pathogenesis of BKD is complex. It is a chronic, multisystemic, granulomatous disease with a number of potential immunomodulatory effects on the host. Given the current limitations for treatment and control of BKD, it is imperative that the pursuit of development of methods of prevention, namely management strategies and vaccination, be continued. To do so, the immunology of BKD must be elucidated in order to better understand and manipulate the associated immune responses to our advantage. This dissertation is composed of four chapters which relate to BKD and the associated immune responses of three species of susceptible salmonid fish as follows: Exogenous stress factors, through stress-induced immunosuppression, have been shown to influence BKD development in cultured salmonids. Chapter 1 examines the effects of two environmental stressors common to fish culture, overcrowding and overfeeding, as they affect BKD development and R. salmoninarumantigen prevalence among juvenile chinook salmon (Oncorhynchus tshawytscha) Immunomodulatory interaction between pathogen and host in BKD is widely reported and merits further investigation. Particularly, the immunological parameters affected and the role of the extracellular protein (ECP) of R. salmoninarum are of interest. Chapter 2 examines the in vivo immune response of rainbow trout (Oncorhynchus mykiss) following exposure to the ECP in terms of both humoral and cell-mediated immunological parameters, including the immune response against another bacterial pathogen. Chapter 3 addresses the in vitro effects of the ECP upon specific splenic immunocyte functions, phagocytosis and respiratory burst activity, in brook trout (Salvelinus fontinalis). The immune-complex mediated hypersensitivity reported to occur with BKD has considerable ramifications for control measures involving immunostimulation via antigen exposure. Further investigation is warranted to discern the significance and consistency of immunological hypersensivity in BKD pathogenesis. Chapter 4 examines the renal lesions, including immunopathologic changes and indications of immune-mediated disease, of brook trout exposed to R. salmoninarum. / Ph. D.

Marine mammals are known for their low susceptibility to mercury toxicity, and it was hypothesized that selenium may play a role in protection against mercury toxicity. To gain insight into the mechanisms of the low susceptibility of cetaceans, we investigated the in vitro effects (1) of mercuric chloride (HgCl₂) on the ultrastructure and cell death of Atlantic spotted dolphin renal cells (Sp1K cells), (2) of HgCl₂ on the cell proliferation and cell cycle status of Sp1K and Rhesus monkey renal cells (MK2), and (3) of sodium selenite (Na₂SeO₃) on cell proliferation and cell death of control and HgCl₂-treated Sp1K cells. HgCl₂ affected multiple organelles and nuclei in Sp1K cells, and induced apoptosis in a time-and dose-dependent manner. Both ultrastructural changes and induction of apoptosis were milder than seen in other cell types in previous publications. In addition, Sp1K cells were able to proliferate at 25 µM HgCl₂ while MK2 cells were killed at 15 µM HgCl₂. An increase in percentage of cells in the G0/G1 phase in the cell cycle and a decrease in S, and G2/M phase cells were seen in Sp1K cells exposed to more than 10 uM HgCl₂ more than 72 hours. MK2 cells showed cell cycle changes only at 24 hours exposure, and may be due to a sensitive subgroup. These data suggested that Sp1K cells were less susceptible than other cell types in a cell-specific way, which was independent of selenium protection. Concurrent exposure to Na₂SeO₃ provided protection against the HgCl₂-induced decrease in cell proliferation of Sp1K. The protective effects were greater if Na₂SeO₃ and HgCl₂ were premixed, but disappeared if exposures did not overlap. Although pretreatments with Na₂SeO₃ alone did not provide protection, they increased the protection of selenium administered later. Furthermore, Na₂SeO₃ decreased HgCl₂-induced apoptosis. These data demonstrated the Na₂SeO₃ protection against HgCl₂ toxicity in Sp1K cells in terms of cell proliferation and apoptosis. This study is the first report that reveals the existence of mercury-selenium antagonism in cultured cetacean cells. The data supported the hypothesis that selenium protection against mercury toxicity is, at least partially, through competition of binding sites and formation of mercury-selenium complex. / Master of Science

Chronic kidney disease (CKD) is a multifaceted health problem with both physical and psychological manifestations. Increased symptom burden is associated with higher risk of mortality, decreased treatment adherence, and impaired quality of life. Despite the recognition of the importance of symptoms, the symptoms and mental health of individuals with CKD remain poorly understood in terms of their measurement, epidemiology and associated service use. The chapters in this dissertation aim to inform these knowledge gaps. Chapter 2 focusses on the symptom burden of patients receiving maintenance hemodialysis treatment and uses exploratory analyses to identify intra-dialytic symptom clusters associated with prolonged dialysis treatment recovery time. Chapters 3 and 4 are population-based studies examining mental health and addictions service utilization in patients with CKD using administrative data in Ontario, Canada. Chapter 3 is a cross-sectional study evaluating the prevalence of individuals with a history of mental health and addiction service use by levels of kidney function. Chapter 4 is a retrospective cohort study evaluating the rates of mental health and addiction service use over time in patients with CKD. Together, these chapters provide further understanding of how symptoms of dialysis and mental health and addiction service use may be measured in this patient population. They also inform considerations for the design of future symptom management and system-level mental health strategies in CKD. / Thesis / Doctor of Philosophy (PhD)