Chronic Kidney Disease (CKD) is common and is associated with increased risk of progression to end stage renal disease, cardiovascular disease and death. CKD is a heterogeneous condition and accurately predicting an individual’s risk for adverse outcomes remains a challenge. Over the past decade there has been a focus on the identification of novel biomarkers that may help improve risk stratification and the prediction of clinical endpoints in this population. The overall aim of this research project was to investigate a series of novel biomarkers in patients from the Chronic Renal Insufficiency Standards Implementation Study (CRISIS), a prospective observational study of outcome in all cause non-dialysis dependent CKD 3-5. The biomarkers selected for this project were Anti-Apolipoprotein A-1 (Anti-apoA-1 IgG), fetuin-A, fibroblast growth factor-23 (FGF23), high sensitivity cardiac troponin T (HS-cTnT), kidney injury molecule-1 (KIM-1), N-terminal pro-brain natriuretic peptide (NT-proBNP), neutrophil gelatinase associated lipocalin (NGAL) and osteoprotegerin (OPG). These biomarkers were chosen to address the three clinical endpoints of progression, cardiovascular disease and death with biomarkers considered both individually and as groups of related markers. The first aim of this project was to examine associations between the novel biomarkers and the clinical characteristics of the CRISIS population. The second aim was to investigate the associations between novel biomarkers and the study endpoints. In the case of FGF23 longitudinal measurements were analysed and in all other cases associations between baseline levels of the markers and clinical outcomes were considered. The third aim was to consider whether the biomarkers investigated in this project actually improve parameters of risk stratification and model discrimination, thereby demonstrating a potential to improve the prediction of outcome events in the CKD population. Many of the biomarkers were independently associated with one or all of the clinical outcomes considered. Despite these associations, it was more difficult to demonstrate clear improvement in risk classification or the prediction of clinical endpoints. Baseline models of standard biochemical and clinical parameters performed very well so even biomarkers that were strongly associated with clinical outcomes resulted in only small incremental improvements in the prediction of outcome events. It is now important to focus on defining how biomarkers may fit into clinical decision pathways.

Chronic kidney disease (CKD) is an important independent risk factor in the development of cardiovascular disease (CVD). Indeed, patients with CKD are far more likely to die from CVD than reach end stage renal disease. Conventional cardiovascular risk factors and co-morbidity contribute to this increased risk of CVD. However, emerging evidence suggests other novel factors including inflammation, oxidative stress, and a shift in the balance of the vasodilator nitric oxide and vasoconstrictor endothelin system, are also important contributors. Despite increasing evidence that the endothelin system plays an important role in the development of CKD and CVD, there has been little research examining possible therapeutic benefits of its modification in patients with CKD. The overall aims of the work presented within this thesis were to examine CVD risk in patients with renal impairment and then to see what impact chronic inhibition of the endothelin system would have on risk factors for CVD and CKD progression. In the first two studies I examined markers of arterial stiffness (AS) and endothelial function in a cohort of patients with immune-mediated renal disease. I was able to show in the acute setting that improvement in renal function following treatment for these conditions leads to significant improvements in AS. Interestingly, in patients who were in remission from their renal disease, only classical cardiovascular risk factors appear to be linked to AS. In the next study I was able to prove that sitaxsentan, a selective oral ETA antagonist, did not cause functional blockade of the ETB receptor in man. This was the first study of its kind to confirm that a “selective” endothelin antagonist truly is selective in vivo: a finding that will allow more accurate mechanistic investigation of the ET system. In the final studies, I showed that in subjects with stable non-diabetic proteinuric CKD, chronic selective ETA receptor antagonism reduces blood pressure and AS, and that these systemic benefits are associated with an increase in renal blood flow and reduction in proteinuria. The reduction in proteinuria is most likely haemodynamic and linked to a fall in GFR and filtration fraction, similar to what is seen with ACE inhibitors. Importantly, these benefits were seen in patients already taking maximally tolerated renin-angiotensin aldosterone system blockade, suggesting that chronic endothelin antagonism could be an important future therapy in the management of CKD. In summary, I have shown that renal impairment can directly affect markers of arterial function and by inference increase the risk of CVD. Chronic antagonism of the endothelin system with ETA receptor blockers would appear to improve many of these biomarkers, including reductions in BP, AS and proteinuria. There were no adverse effects reported in these studies, suggesting that selective ETA antagonism may be safe enough for clinical development in CKD patients. Further larger clinical trials are warranted.

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Thromboxane receptor (TPr) activity is elevated in diabetes and contributes to complications of diabetic kidney disease (DKD). TPr blockade appears to have therapeutic potential. Several rodent models of DKD show attenuation of renal damage and proteinuria upon administration of the TPr antagonist, S18886. However, the cellular targets that underlie the injurious effects of TPr activation in DKD remain to be elucidated. A pilot study in our laboratory subjected a conditionally-immortalized mouse podocyte cell line to high glucose (25 mM D-glucose) and equibiaxial mechanical stretch (an in vitro simulator of increased glomerular capillary pressure associated with glomerular hyperfiltration in early diabetes). qRT-PCR revealed that exposure of podocytes to mechanical stretch (10% elongation) and high glucose for 6 hours yielded a 9-fold increase in TPr mRNA levels vs. controls (non-stretch, 5mM D-glucose + 25mM L-glucose) (p<0.05, n=5). We hypothesized that TPr expression and activity are increased in podocytes during the onset of DKD resulting in maladaptive effects on this key glomerular filtration barrier cell type. We showed that enhanced TPr signaling threatens podocytes viablility. Cultured podocytes treated with the TPr agonist, U-46619 (1 μM) for 24 hours are more vulnerable to apoptosis as quantified by Hoescht 33342 (20% cell death p<0.001, n=3) , TUNEL (30-fold increase, ns, n=3) and Annexin-V labeling (3-fold increase, p <0.001, n=3). To further support these in vitro findings, we developed a transgenic mouse with podocyte-specific overexpression of TPr. A construct consisting of a desensitization resistant mutant of the human TPr with both N- and C-terminal HA-epitope tags under the control of an 8.3 kb fragment of the immediate 5’ mouse NPHS1 promoter was cloned, isolated and injected into FVB/n oocytes that were implanted into pseudopregnant CD1 females. Founders were characterized for TPr transgene expression, and TPr transgene mRNA levels were detected by qRT-PCR. Our in vitro results suggest that increased TPr expression in podocytes of diabetic mice may contribute to filtration barrier damage and have important implications in the development and progression of DKD.

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Background and objectives: Chronic kidney disease-mineral and bone disorder (CKD-MBD) is a systemic condition defined by an increase in cardiovascular calcifications and bone fragility. The condition is diagnosed by abnormal serum concentrations of calcium, phosphorus, parathyroid hormone and vitamin D. These biochemical abnormalities have been linked to abnormal bone metabolism as well as cardiovascular calcifications if left untreated. Phosphate binders are known to cause phosphate reduction through mechanisms involve the gastrointestinal route. Their relative effects remain uncertain. Controversy arises because of concerns related to systematic effects, tolerability, costs and impact on patient important outcomes. The objective of Chapters 2 and 3 was to explore the relative effectiveness of phosphate binders on patient-important outcomes and laboratory outcomes in patients with CKD-MBD using the frequentist and Bayesian approaches, respectively. The purpose of Chapter 4 was to critically appraise clinical practice guidelines addressing CKD-MBD. Methods and results Chapter 2: We performed network meta-analyses for all cause-mortality for individual agents (seven-node analysis) and conventional meta-analysis of calcium vs. non-calcium based phosphate binders (NCBPB) for all-cause mortality, cardiovascular mortality and hospitalization. Our results suggested higher mortality with calcium than either sevelamer in our network meta-analysis or NCBPB in our conventional meta-analysis. Conventional meta-analysis suggested no statistically difference in cardiovascular mortality between calcium and NCBPBs. Chapter 3: We performed Bayesian network meta-analyses to calculate the effect estimates (mean differences) and 95% credible intervals for serum levels of phosphate, calcium and parathyroid hormone. Moderate-quality evidence suggests superior effect of active treatment categories as compared to placebo for reducing serum phosphate. Our NMA results did not find statistically significant difference between active treatment categories in lowering serum phosphate. Chapter 4: We performed a systematic survey to critically appraise clinical practice guidelines addressing CKD-MBD. Most guidelines assessing CKD-MBD suffer from serious shortcomings using the Advancing Guideline Development, Reporting and Evaluation in Health Care instrument II (AGREE) criteria; a minority, however, fulfill the criteria. Limitations with respect to AGREE criteria do not, however, necessarily lead to inappropriate recommendations. Conclusion: Given the likely mortality reduction with sevelamer versus calcium, the results suggest that higher calcium levels associated with calcium based phosphate binders may contribute to the mortality differential. We found that most clinical practice guidelines related to CKD-MBD were not satisfactory with major problems with rigor, update and implementation. Recommendations were consistent and thus unassociated with guideline quality. In other instances, however, this may not be the case, and ensuring trustworthiness of guidelines will require adherence to methodological standards. / Thesis / Doctor of Philosophy (PhD)

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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.

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.