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The complex interplay between regulatory factors of kidney mineral ion homeostasis are difficult to investigate in vivo. In addition, preclinical drug safety testing is limited by lack of translational tools for screening novel drugs for nephrotoxicity. At Cardiff University, my PhD project aimed to characterise tools to address these needs. At AstraZeneca, the use of tissue biomarkers as tools for assessing kidney injury in retrospective studies where urine biomarker samples are not available, was evaluated. To enable studies of mineral ion homeostasis, an in vitro human primary renal cell model was characterised. Cells expressed phenotypic marker proteins (E-, N-Cadherin) as well as proteins involved in mineral ion homeostasis (FGFR1-4, Klotho, NaPi IIa, NCX1, PMCA1). They also exhibited morphological features and functional characteristics of renal cells in vivo. Data were presented as an oral abstract communication at the American Society of Nephology Meeting, 2013. To enable studies of drug-induced nephrotoxicity, in vitro primary mouse and human renal cell models were characterised. A concentration-dependent increase in apoptosis was observed in mouse cells following 24-hour treatment with medium containing cisplatin, cyclosporin A and BEA. Following 24-hour treatment, the same nephrotoxins increased expression of the kidney injury biomarkers kidney injury molecule-1 (KIM-1) and osteopontin. Human cells responded to 24-hour treatment with medium containing cisplatin with expression of KIM-1, osteopontin and clusterin. Parallel use of such rodent and human models would revolutionise preclinical translational drug safety assessment. Cisplatin-treated rats were used to evaluate the use of tissue biomarkers as tools in studies where urine biomarker samples are not available, such as retrospective studies in drug safety testing. Of the biomarkers investigated, KIM-1 and osteopontin showed greatest correlation with their corresponding urinary biomarkers. As such, they offer greatest utility as tissue biomarkers in retrospective studies. Data were published in Toxicologic Pathology (Wadey et al. 2013).

The best characterised collagen IV disease is Alport syndrome, caused by α3.α4.α5(IV) mutations. Mutations affecting α1(IV)2α2(IV) can cause diseases, such as haemorrhagic stroke, aneurysm, haematuria, proteinuria, cataracts, vascular tortuosity and iris hypoplasia. Recent data has shown that α and COL4A2 mutations may be more common in the population than previously anticipated. However, the mechanisms of disease of all collagen IV mutations are poorly understood. The aim of this thesis was to investigate the disease mechanism in a selection of COL4A1, COL4A2 and COL4A5 mutations. Collagen IV is folded in the endoplasmic reticulum (ER) then secreted to provide the major structural component of the basement membrane (BM). As such, a combination of ex situ biopsies, in vitro cell cultures and an in vivo mouse model provided an excellent platform for investigating the intracellular and extracellular effects of mutant trimers. Primary dermal fibroblasts containing COL4A2 and COL4A5 mutations, and a Col4a1 mouse model were treated with the FDA approved chemical chaperone phenyl-4-butyric acid (PBA) to determine its effects on intracellular ER retention of trimers (which can activate ER stress), extracellular trimer incorporation into the BM, and disease phenotypes. In vitro and ex situ investigation of a COL4A2 mutation suggested that intracellular α1(IV)2α2(IV) accumulation and ER stress associated with severe BM defects and the disease. Intracellular accumulation and ER stress were reduced by PBA treatment, and treatment of a Col4a1 mouse model displayed elevated α1(IV)2α2(IV) incorporation onto the BM. In vivo PBA treatment did not exacerbate any defects, rescued some disease phenotypes, and had no effect on others. In addition, one of five COL4A5 mutations analysed in vitro displayed intracellular α3.α4.α5(IV) accumulation and ER stress that were ameliorated by PBA. Collectively these data suggest that ER stress and/or reduced α1(IV)2α2(IV) BM incorporation underlie some collagen IV pathologies, and provide novel evidence that they may be amenable to chemical chaperone therapy. More investigation is now needed to further dissect the relative contributions of ER stress and BM defects to collagen IV diseases.

Chronic kidney disease (CKD) is associated with an increased risk of cardiovascular disease and end stage renal failure. Accurate identification of those with a reduced glomerular filtration rate and significant proteinuria facilitates early diagnosis and risk stratification. This thesis explores the optimal measure of proteinuria, to accurately quantify proteinuria and as a predictor of renal and patient outcomes. We examine the prevalence of CKD in a general population cohort and assess the impact of different estimated glomerular filtration rate (eGFR) formulae. We explore the prognostic role of reduced eGFR and proteinuria in patients with hypertension and present the baseline characteristics of a community cohort study of patients with predominantly early CKD. They will be followed for ten years to identify predictors of cardiovascular and renal outcome. Urine total protein:creatinine ratio (TPCR) and albumin:creatinine ratio (ACR) have largely replaced 24-hour urine collections for proteinuria quantification. The performance of these spot measures to identify significant proteinuria is compared in a cohort of 6842 patients attending a general nephrology clinic. Both tests perform well overall but TPCR is statistically significantly superior as a predictor of 24-hour total proteinuria than ACR (as measured by the area under the receiver operator characteristic (ROC) curve to predict 1g/day total proteinuria). On sub-group analysis the performance of the spot samples is poorer in women and the elderly, likely as a result of low muscle mass and low urine creatinine (the denominator in TPCR/ACR). The performance of TPCR and ACR were then compared as predictors of outcome in a similar cohort of 5586 CKD patients using a hierarchical Cox survival model. TPCR and ACR both performed well as independent predictors of death, commencement of renal replacement therapy (RRT) and doubling of serum creatinine. Notably TPCR performed well at low levels where albuminuria has been considered superior. These findings are novel. The spot samples performed as well as 24-hour collections in the sub-group with timed urine collections. The National Institute for Health and Clinical Excellence in England recommend ACR to monitor all patients with CKD; the Scottish Intercollegiate Guidelines Network recommend TPCR for non-diabetic renal disease. Therefore, we investigated the implications of these recommendations using survival modelling. The same cohort was divided into 5 groups: no proteinuria, low proteinuria (using TPCR and ACR), high proteinuria (TPCR and ACR) and two groups where TPCR and ACR were discordant (i.e. TPCR above the diagnostic threshold but ACR below it and vice versa) using the recommended thresholds (ACR 30mg/mmol/TPCR 50mg/mmol to predict 0.5g/day total proteinuria and ACR 70mg/mmol/TPCR 100mg/mmol to predict 1g/day total proteinuria). Using univariate survival analysis the discordant group had significantly poorer outcomes (using the same outcomes as previously) than those with significant proteinuria as measured by both tests. The discordant group was older with poorer renal function and some of the excess risk was abolished on multivariate analysis, however the risk did not return to the level of those without detectable proteinuria. TPCR, but not ACR, measures non-albumin proteins and these may have pathophysiological roles in progression. This requires further study. However this analysis confirmed that TPCR identifies patients at high risk of adverse outcomes. TPCR and ACR may vary as a result of muscle mass. We adjusted TPCR and ACR for estimated creatinine excretion (ECE) (calculated using the Cockcroft and Gault formula) and performed cross-sectional and longitudinal analyses. Adjusting TPCR and ACR for ECE improves prediction of significant proteinuria in sub-groups with poor baseline test performance (such as women and the elderly) using ROC curve analysis. However when adjusted and unadjusted values were compared as predictors of outcome (using a net reclassification index analysis) adjusted values were significantly inferior. Urine creatinine is an independent predictor of mortality and hence may be directly contributing to the predictive value of TPCR and ACR rather than simply correcting for urine flow rate. As such, adjusting for ECE may act to remove the effect of a second independent predictor, leading to inferior test performance. Therefore the decision to adjust TPCR and ACR for ECE depends on the test application: to predict significant proteinuria adjustment of TPCR and ACR is of benefit, but adjustment leads to inferior performance as a prognostic test. The prevalence of CKD stages 3-5 was assessed using a general population laboratory database. Overall population prevalence was 5.63% using the modification of diet in renal disease (MDRD) formula and fell to 4.94% when the CKD-Epidemiology group (CKD-EPI) formulae were applied. Those reclassified to an earlier stage of CKD were predominantly middle aged women. Prevalence over a five year period was found to be stable using the CKD-EPI formulae but rose slightly according to MDRD. Proteinuria and eGFR were assessed as predictors of outcome in a large specialist hypertension clinic cohort. On multivariate survival analysis both baseline dipstick proteinuria and an eGFR<60ml/min/1.73m2 remained strong independent predictors of cardiovascular and all-cause mortality, despite intensive specialist intervention to control blood pressure. These simple tests should be advocated for risk stratification in these patients. Lastly the baseline characteristics of a community CKD cohort are presented. We recruited 411 participants from seven general practices around Ayrshire and a detailed baseline clinical and biochemical assessment was performed. Patients were invited to participate if they were included in the primary care register of CKD stages 3-5. Over a quarter had an eGFR>60ml/min/1.73m2 on the meat-fasted study sample. Proteinuria was of notably low prevalence and the cohort had a large burden of cardiovascular disease. Complications of renal disease were uncommon. The characteristics of the cohort differ from those under hospital follow-up. Their long term outcomes should contribute to refining risk stratification in this population. Proteinuria and eGFR are key aspects of diagnosis and monitoring in CKD. Identification of the optimal measures of both is essential and findings presented here contribute to that. There is a need to refine risk stratification in CKD, to identify those who require intensive intervention, and to reassure the rest. The findings of this thesis also contribute to that. Further study is required to refine the core aspects of diagnosis and investigation of CKD.

Chronic kidney disease is common and associated with an elevated cardiovascular risk, as well as the long-term risk of renal failure. At present, therapeutic approaches to managing chronic kidney disease (CKD) do not fully reverse these risks. This has led to study of the determinants of pathological outcomes in these patients, with the hope of further therapeutic interventions to reduce these risks. Mineralocorticoids, predominantly aldosterone, are produced by the adrenal cortex and have a vital role in maintaining sodium status and blood pressure. However, high levels of aldosterone in humans are known to produce an adverse phenotype of hypertension and a disproportionately elevated cardiovascular risk. Furthermore, in animal models of renal failure, elevated aldosterone levels stimulate renal damage, in the presence of a high sodium milieu. These laboratory findings have been translated to provide a basis for several short-term follow-up clinical trials looking at the impact of non-genomic non-natriuretic doses of mineralocorticoid receptor inhibition in patients with chronic kidney disease. These studies have shown a reduction in proteinuria, often independent of decline in blood pressure. However, there is a paucity of baseline physiological data relating to the normal regulation of mineralocorticoid synthesis and action in chronic kidney disease. The response of the adrenal cortex to renal failure is not understood. Is mineralocorticoid synthesis regulated in the usual way? Are the stimulators of mineralocorticoid production and release affected by uraemia? Is dietary sodium intake associated with steroid status and adverse outcomes in humans? The hypothesis of this thesis was that the renin-angiotensin-aldosterone system is inappropriately activated in patients with chronic kidney disease. Secondly, that high levels of mineralocorticoids are associated with adverse end-organ damage including proteinuria excretion, left ventricular hypertrophy, endothelial dysfunction, elevated pulse wave velocity and markers of renal fibrosis. Furthermore, that these deleterious effects are associated with sodium status and that an elevated dietary sodium intake is independently associated with increased renal and cardiovascular risk. In order to test these hypotheses, 70 patients with CKD and 30 patients with essential hypertension (EH) were recruited and underwent detailed clinical and biochemical phenotyping. This included 24 hour urinary steroid metabolite analysis, plasma renin and aldosterone measurement, cardiac magnetic resonance imaging, carotid-femoral pulse wave velocity and assessment of endothelial function. 20 It was shown that levels of the main mineralocorticoids (MC) (aldosterone and deoxycorticosterone) are not elevated in patients with CKD, as compared with patients with essential hypertension (EH). However, the determinants of levels of MC excretion differed between the two conditions. In CKD, excretion of MC metabolites was directly proportional to excretion of urinary sodium. A high urinary sodium (a marker of dietary sodium intake) was associated with a higher excretion of tetrahydroaldosterone (THALDO - the main aldosterone metabolite). In patients with EH, no relationship was seen between urinary steroid excretion and urinary sodium excretion. This is a novel relationship between the kidney and adrenal gland which questions the conventional wisdom that the adrenal cortex is unaffected by uraemia and prompts further study into the regulation of steroid synthesis in CKD. Furthermore, it was shown for the first time that 24h excretion of tetrahydrodeoxycorticosterone (THDOC) is an independent predictor of left ventricular mass index and that THALDO is an independent predictor of proteinuria excretion – demonstrating a relationship between mineralocorticoids and two of the main predictors of mortality in CKD. An interaction between sodium, MCs and these two features was also demonstrated. No association between levels of mineralocorticoids and vascular function was seen. Urinary 24 hour excretion of sodium was significantly associated with endothelial dysfunction in patients with CKD and pulse wave velocity in patients with essential hypertension. Retrospective data analysis further confirmed an association between a high dietary sodium intake and adverse outcomes. In a study of 498 patients with CKD and a median follow-up of 7 years, an elevated 24h urinary sodium to creatinine ratio was shown to be associated with an increased risk of death. There was however no independent association with renal progression or requirement for renal replacement therapy. This is the first time that sodium intake has been clearly linked to adverse outcomes in patients with CKD. Lastly, laboratory work demonstrated that steroid stimulation (aldosterone or cortisol) of human proximal tubular cells resulted in increased collagen 1 gene expression, but only in the context of a high sodium environment. Collagen 1 is deposited in renal interstitial fibrosis. This effect was inhibited by MR blockade, further expanding on the potential role 21 of steroids in the progression of CKD and again confirming the relationship between salt and steroids. In conclusion, in this thesis it has been demonstrated that production of MCs in patients with CKD is closely associated with urinary sodium excretion (a surrogate for dietary sodium intake). This relationship is novel and not seen in patients with essential hypertension. It suggests that the response of the adrenal cortex in the context of uraemia is altered. Moreover, levels of mineralocorticoids are independently associated with left ventricular mass index and proteinuria excretion, both significant predictors of mortality, in patients with CKD. Dietary sodium intake has been shown to be an independent predictor of mortality and laboratory studies have demonstrated that mineralocorticoid receptor binding in a high sodium environment is associated with collagen 1 gene upreguation. These findings have important implications for the role of adequate renin-angiotensin-aldosterone blockade in patients with CKD and suggest that the addition of a mineralocorticoid receptor blocker and dietary sodium restriction should be advocated.

Background. Native arteriovenous fistula (AVF) is the vascular access of choice and its use c.f. catheters is associated with sustained reduction in mortality. This may be due to factors beyond dialysis catheter associated sepsis. This study aims to investigate the impact of AVF formation on the spectrum of cardiovascular factors that might be important in the pathophysiology of cardiovascular diseases in CKD patients. Methods. We recruited 43 pre-dialysis patients who underwent AVF formation. Patients were studied two weeks prior to AVF operation, two weeks and three months postoperatively. Haemodynamic variables were measured using pulse wave analysis, carotid femoral pulse wave velocity (CF-PWV) by applanation tonometry and AVF blood flow by Doppler ultrasound. Bioimpedence analysis was performed and patients underwent serial transthoracic echocardiography. Laser Doppler Perfusion Imaging and iontophoresis were used to assess endothelial dependant (ED) and non-endothelial dependant (NED) vasodilatation. Results. AVF formation was successful in 30/43 patients. Two weeks postoperatively, total peripheral resistance decreased (-17 18%, p=0.001), stroke volume tended to rise (12 30ml, p=0.053) and both heart rate (4 8bpm, p=0.01) and cardiac output (1.1 1.5l/min, p=0.001) increased. Systolic and diastolic blood pressures reduced (-9 18mmHg; -9 10mmHg; ≤ p=0.006). CF-PWV reduced (-1.1 1.5m/sec, p=0.004). Left ventricular ejection fraction (LVEF) increased (6 8%, p<0.001). Patients with successful AVF formation had a significantly reduced ED vasodilatation in the fistula arm -36±46%, p<0.001. Only NED vasodilatation was significantly reduced in the non-fistula arm 23±40%, p=0.01. Patients who had unsuccessful AVF operation exhibited no recordable changes. All the observed haemodynamic changes were largely maintained after 3 months. No change in hydration status/body composition was observed. AVF formation resulted in a sustained reduction in arterial stiffness and BP as well as an increase in LVEF. Furthermore, there were significant changes in the local and systemic microcirculation. Overall, post AVF adaptations might be characterised as potentially beneficial in these patients and supports the widespread use of native vascular access, including older or cardiovascular compromised individuals.

Human epidemiological studies have indentified obesity as an independent risk factor for renal disease. In addition, maternal nutrient restriction (MNR) during gestation results in a series of adaptations that may predispose those offspring to obesity and hypertension. Recent reports demonstrated that obese sheep exposed to MNR during early to mid gestation have a predisposition to ectopic lipid deposition in the heart and a rise in necrotic adipocytes, which are markers of severe metabolic dysfunction. Surprisingly, in this model of MNR, the renal tissue of those offspring showed an apparent reduction in cell apoptosis. However, renal diseases associated with obesity have a slow progression and their mechanisms are not completely understood. In the light of these results, the main hypothesis of my thesis is that the renal amelioration observed in those nutrient restricted (NR) obese offspring is a product of post-injury responses, inducing scarring and others adaptations to obesity. Therefore, some of the main regulatory factors in renal and perirenal adipose tissue (PAT) development were analysed in seven-day-old and one-year-old obese sheep offspring exposed to MNR (3.5 KJ/days) from 30 to 80 gestational days (term ≈ 145 days). At one week of age, the renal composition and gene expression showed small changes between NR offspring and those born to control mothers. However, in PAT of NR offspring, an increased in expression of the methyltransferase DNMT-1 and a decrease in mRNA abundance of IGF-2 were observed. At six months of age, obesity onset was accompanied by raised plasma cortisol and leptin concentration in NR offspring compared to control. By one year of age, whilst plasma leptin concentration was similar between the obese groups, in the PAT of the NR offspring there was an increase in gene expression of pro-inflammatory factors and DNMT-1, suggesting advanced adipose tissue remodelling. In kidney, regardless of in utero diet, obesity induced similar amounts of oxidative stress, activation of cellular proliferative factors and collagen deposition. Although, both obese groups had equal activation of pro-apoptotic factors (e.g p-53 and Bax), renal iron and mRNA abundance of the death receptor, Fas only increased in obese offspring born to control fed mothers. A major finding in the NR kidney was increased ectopic triglyceride deposition, indicating early onset post-injury in response to sympathetic activation and lipotoxity. The main conclusion of my thesis is that functional changes observed in the adipose tissue lead the kidney to an initial cycle of cell proliferation, apoptosis and arrest, followed by tissue remodelling, characterised by the presence of collagen. However, this adaptation to obesity is accompanied by an increase in lipid deposition in the kidney of the NR group that may be a sign of an advanced state of metabolic dysfunction.

The primary objective of this thesis was to determine any associations between herbal and dietary supplement (HDS) use and the fast progression of chronic kidney disease (CKD) in Thai outpatients with CKD. The secondary objectives were to determine any associations between HDS use and CKD complications, and the prevalence and reasons for HDS use. A survey recruited 421 outpatients with stages 3 to 5 CKD from two kidney clinics in Thailand, from January to June 2012. A prospective cohort study followed up these respondents, in particular noting their serum creatinine, as well as serum levels of potassium and phosphate, for 12 months. Such data were extracted from patients’ medical notes. Three hundred and fifty-seven respondents were followed up. The exposed group was defined as the current and regular users of HDS, and the primary outcome of the cohort study was defined as either a decline in the estimated glomerular filtration rate of at least 5 ml/min/1.73m2/year or the initiation of renal replacement therapy. Sixteen HDS users were recruited from the survey to be interviewed about their reasons for using HDS, using open-ended questions to elicit information in the qualitative study. Exclusion criteria were those with had received renal replacement therapy before recruitment. Univariate and multivariate analyses were performed to determine the associations using Chi-squared tests and multiple logistic regressions. Tests were 2-tailed and a p-value < 0.05 was considered statistically significant. The prevalence of HDS use during the previous year in Thai patients with CKD was 45% (95%CI 40%-50%). The most frequently reported influences on HDS use in the survey and the qualitative study were family members, friends and perception of benefits gained from using HDS. An association between HDS use and CKD progression was not found (adjusted OR 1.16, 95%CI 0.66 – 2.03). Two respondents (0.6%) had acute kidney injury, which may be related to the use of unknown Chinese herbal medicines or river spiderwort combined with diclofenac; issues which were reported by their doctor in their medical note. HDS use was associated with uncontrolled hyperphosphatemia (adjusted OR 3.53, 95%CI 1.20 – 10.43), possibly due to the HDS used in the cohort study which contained phosphate or vitamin D. Health care providers should closely monitor CKD patients using Chinese herbal medicine, river spiderwort or HDS containing phosphorus or vitamin D. Further studies need to examine renal adverse effects of specific herbal medicines, particularly in relation to acute kidney injury.

Monoclonal immunoglobulin free light chains (FLCs) cause a range of disorders in the kidney. In multiple myeloma, FLCs can activate the proximal tubule to release MCP-1, an important cytokine in renal fibrosis. Distal tubular cast formation can also occur when FLCs co-precipitate with uromodulin. However a pathogenic role for the elevated polyclonal FLC concentrations seen in chronic kidney disease has not been assessed to date. This thesis explores the biology of monoclonal FLCs as well as polyclonal FLCs. Detailed histological analyses demonstrated that in multiple myeloma, interstitial fibrosis can progress rapidly in situ and indicated that intratubular cast numbers might be linked to potential for renal recovery. The functional basis of this fibrosis was explored by in vitro studies, which showed that upon endocytosis of FLCs, oxidative stress activated redox signalling, resulting in MCP-1 production. Further in situ analyses showed that in chronic kidney disease, polyclonal FLCs co-localised with uromodulin in distal tubular casts. Relationships between these casts and markers of progression of chronic kidney disease were demonstrated. In vitro analyses then showed that polyclonal FLCs bind to uromodulin and promote aggregation. These findings: (i) further delineate the pathways for proximal tubular injury in myeloma and (ii) indicate a potential pathogenic role for polyclonal FLCs in the distal nephron.

Chronic kidney disease affects 10-13% of the population. The dominant processes that promote kidney disease, irrespective of the trigger, occur in the stromal compartment where fibrosis is considered the hallmark of progressive renal disease. Recent studies have highlighted the importance of the vasculature and the role of the renal pericyte as a progenitor of activated matrix-depositing stromal myofibroblasts, cells that drive the development of renal fibrosis. CD248 is a 175 KDa type I transmembrane glycoprotein expressed at low levels in non-inflamed kidney by resident renal stromal cells (pericytes and myofibroblasts). In this thesis I demonstrate that CD248 expression is increased in a cohort of patients with progressive renal fibrosis (n=93). Furthermore, increased CD248 expression in the kidney stroma is an independent risk factor for the progression of renal disease. I have then used an established murine model of renal fibrosis (unilateral ureteric obstruction) to characterise the origin, phenotype and function of CD248+ cells in vitro and in vivo. A transgenic mouse, with a targeted disruption to the CD248 gene has been used to assess the causal role that CD248 plays in the pathogenesis of renal fibrosis. Mice deficient in CD248 are protected against myofibroblast accumulation, tissue fibrosis and microvascular rarefaction following renal injury. In vitro data suggests that this phenotype may be due to a defect seen exclusively in stromal cell, but not epithelial cell, function as a consequence of the loss of CD248. Taken together these studies suggest that CD248 represents a novel stromal cell specific target for the treatment of chronic kidney disease.