Introduction. Connective tissue growth factor (CTGF) is the only CCN member recognized as a mediator of chronic fibrotic disease. Accumulating evidence suggests that CTGF is important as a downstream effector of transforming growth factor (TGFβ) in modulating a sustained pro-fibrotic signal in in vivo models. Moreover, in cultured human proximal tubule epithelial cells (PTECs) CTGF mRNA is readily upregulated by TGFP and the nascent protein accumulates extracellularly. How pro-fibrotic signalling occurs is yet to be ascertained but it likely involves extracellular interactions with secreted CTGF. The tetra-modular structure of CTGF has been shown to possess multiple binding sites for ligands present in the extracellular milieu including matrix proteins, growth factors including the TGFβ superfamily and cell surface receptors. As such, CTGF can behave as a bridge between matrix and cell. Moreover, individual modules possess intrinsic biologic activity. Materials and Methods. Experiments were performed in cultured renal human renal epithelial cells. Recombinant human CTGF protein was generated in-house following plasmid transfection into context relevant PTECs. Immunoblotting and ELISA techniques were used to investigate protein expression in response to incubation with study protein in isolation or in combination with TGFP superfamily members. Protein-protein binding was investigated using surface plasmon resonance (SPR) in order to elucidate how CTGF might regulate TGFβ superfamily cell signalling. Additional experiments with an alternative CCN member, CCN3, were performed. Results. Both full-length and C-terminal CTGF bind to TGFβ and BMP7 and in human renal epithelial cells, this binding modulates the downstream Smad signalling pathways associated with fibrosis. In cultured human podocytes, CTGF drives TGFβ-dependent signalling in the absence of exogenous TGF(3 suggesting activation of latent TGFβ. Moreover, C-terminal CTGF increases the generation of pro-fibrotic markers aSMA and fibronectin, both of which are subsequently blocked by inhibiting the TGFp receptor. Unlike CTGF, an alternative CCN member CCN3 reduces TGFβ-induced signalling in PTECs. Conclusion. CTGF protein has multiple binding sites and modulates the cellular responses of TGFp superfamily members on human renal epithelial cells. Both full-length and C-terminal CTGF bind to TGFβ and BMP-7. CTGF also appears to bind to the receptors of the TGFβ superfamily. C-terminal CTGF has intrinsic profibrotic activity that differs from the full-length protein as demonstrated in cultured human podocytes. The pro-fibrotic activity is suppressed by CCN3 in the CTGF rich environment of the cultured PTEC. The interplay of different CCN proteins in modulating fibrotic pathways in renal epithelial cells warrants further investigation.

Infertility is a significant life event affecting around one in seven couples in the UK. The development of Assisted Reproductive Technologies (ARTs) such as In Vitro Fertilisation (IVF) have encouraged the idea that infertility can, and should, be treated. By seeking medical attention to overcome this condition, couples are understood to have begun an ‘infertility journey’. The study aim was to investigate the experiences of couples starting infertility investigations and treatments. Using an ethnographic methodology with a longitudinal design, involving iterative rounds of observation and interviews with the same participants. The findings show couples were resistant to becoming fertility patients and starting treatments, and this was often connected to the meaning that infertility had for them. The discomfort and challenge of this transition, previously described as ‘mazing’, was not always recognised or fully appreciated by the healthcare staff involved with the couples. ARTs are becoming ubiquitous and this has increased the mazing undertaken by couples because of the necessity for multiple treatment cycles. The commercialisation of fertility treatments, often including payment for cycles, is also unhelpful for couples’ decision-making. Couples are jointly involved in negotiating treatments and future planning, but clinics focus almost exclusively on the female partner. The ‘journey’ retains its open-ended quality because treatments, belying their promise and hope, do not usually resolve the uncertainty of infertility. Conclusion Treatments for infertility, including ARTs, are portrayed as straightforward, however this study finds that couples, particularly during the early stages from first GP referral, are reluctant to engage with medical fertility pathways and do not find the processes ‘routine’. By acknowledging this, and showing an interest in the personal and social context of their patients’ infertility, clinical staff can demonstrate they are listening to their patients. Couples may find this altered communication helps improve their experiences of fertility treatments.

The overarching aim of this PhD thesis is to develop methods, which will ultimately improve the management of patients with acute kidney injury (AKI). Over the years one inherent problem in both diagnosing AKI clinically and reviewing and comparing studies published in the literature has been the numerous definitions used to define AKI. 87 With now accepted definitions of AKI, the first question raised was to determine the true impact of AKI, in terms of incidence and outcomes, for both the patient (morbidity and mortality) and the healthcare economy. A retrospective observational database study was performed from secondary care in East Kent (adult catchment population of 582,300). All adult patients (18 years or over) admitted between 1st February 2009 and 31st July 2009, were included. Patients receiving chronic renal replacement therapy (RRT), maternity and day case admissions were excluded. AKI was defined by the acute kidney injury network (AKIN) criteria. A time dependent risk analysis with logistic regression and Cox regression was used for the analysis of in-hospital mortality and survival. The incidence of AKI in the 6 month period was 15,325 pmp/yr (adults) (69% AKIN1, 18% AKIN2 and 13% AKIN3). In-hospital mortality, length of stay and ITU utilisation all increased with severity of AKI. Patients with AKI had an increase in care on discharge and an increase in hospital readmission within 30 days.  In comparison with patients with no AKI those with AKI stage 1 had a 52% longer length of stay (LOS) in hospital, a 2.8-fold increased risk of admission to the intensive therapy unit (ITU), a 39% longer ITU stay (in those who went to ITU), and a 2.4-fold greater in-hospital mortality. Furthermore, patients with AKI stage 1 had twice the long-term risk of death, a 33% higher likelihood of an increase in care, and a 42% higher risk of re-admission within 30 days. In those patients with AKI stage 3 (the subject of the NCEPOD report) 100 hospital LOS doubled, there was a 22 times higher risk of admission to ITU and ITU LOS was also doubled, consistent with national data from the Intensive Care National Audit and Research Centre. A further study using this data in collaboration with Marion Kerr (health economist) at the Department of Health, suggested the annual number of excess inpatient deaths, with AKI in England may be greater than 40,000, 106 and the annual cost of AKI-related inpatient care in England is estimated at £1.02 billion. 106 With the problem now evident and clearly defined, the first stage in improving management was to alert clinicians to the presence of AKI as soon as possible to allow early recognition and intervention. Here the development of a static AKI alert report delivered to the critical care outreach team and specialist renal team is documented. A qualitative analysis was then used to explore the effect of professional interactions, information sharing, and personal and professional characteristics on the use of electronic clinical information and clinical decision support. Key areas highlighted in the qualitative analysis included real-time delivery of AKI alerts, clear responsibility of care to be with the clinical teams with advice from the critical care outreach nurses and renal consultants as required, and improved communication with the clinical teams looking after the patients. This work informed a development partnership with a commercial company (Careflow Connect Limited) to deliver real-time alerting of acute kidney injury to clinicians at the point of care and allow collaboration within the clinical team and also with the specialist renal and critical care outreach teams. However, in any disease process, while we can optimise our measures in place (as above) to alert to the presence of a disease (in this case acute kidney injury (AKI)) and manage it effectively and efficiently at recognition, the ultimate form of treatment is the prevention of the disease occurring in the first place. Hence, in order to achieve this we need to determine the patient at risk. Firstly, potential risk factors were explored. Three time points were also defined where significant clinical decision making takes place and at which points the use of risk models would have greatest impact on clinical care and patient management. These were the point of admission to hospital to guide renal function testing and inform admission planning, and secondly, at 24 hours after admission, often on the post-take ward round to highlight patients who are likely to develop new or worsening AKI if already present, in the first 72 hours of hospital admission so that appropriate management decisions can be made on the ward round. The study population included hospital admissions to the three acute hospitals of East Kent Hospitals University NHS Foundation Trust (EKHUFT) in 2011, excluding maternity and elective admissions. For validation in a second population the study included hospital admissions to Medway NHS Foundation Trust. The study developed and assessed traditional methods to provide risk models for the prediction of new or worsening AKI in patients presenting to hospital and in their management within the first 24 hours of admission. Ordinal logistic regression with uni-variable analyses were used to inform the development of multi-variable analyses. Backward selection was used to retain only statistically significant variables in the final models. The models were validated using actual and predicted probabilities, Area Under the Receiver Operating Characteristic (AUROC) curve analysis and the Hosmer Lemeshow test. The analysis identified key variables which predict AKI both at admission and 72 hours post admission. Validation demonstrated area under ROC of 0.75 and 0.68 respectively. Predicting worsening AKI during admission was unsuccessful. These models were also re-defined with use of the NHS England algorithm to define AKI which produced similar results with area under ROC of 0.73 and 0.67 respectively. The work reported here has demonstrated the significant morbidity and mortality both long and short term of patients who experience acute kidney injury managed in hospital and has developed methods of alerting the presence of AKI to the point of care in real-time to ensure efficient intervention with an aim to improve these outcomes. Qualitative work has also highlighted the complexity regarding the implementation and delivery of alerting systems to the clinical front line. The work reported in this thesis has also demonstrated that routinely available data can be used to highlight patients at risk of acute kidney injury both at the point of admission to hospital and following admission.

The aim of this thesis is to evaluate the extent to which cost-effectiveness of a recently launched drug in the treatment of overactive bladder is supported by real world evidence. The demand for real world effectiveness data by Payers and decision-makers is increasing to better manage the uncertainty at the time of making reimbursement decisions. Real world data can help to fill the knowledge gap between clinical trials and actual clinical practice. Overactive bladder is one of many chronic conditions likely to impact people as they age. It is a common condition characterised by a group of lower urinary tract storage symptoms, which has a profound and measurable negative effect on patient health-related quality of life (HRQoL). The economic burden of OAB is also considerable. Antimuscarincs have been the mainstay pharmacological treatment for OAB for over 30 years, but adverse events and persistence on medication remain a key issue. Mirabegron, a ß3 adrenoceptor agonist provides an alternative option. The thesis presents and critiques 9 peer-reviewed publications to demonstrate the value evidence generated pre-health-technology assessment (HTA) and that generated post-health -technology assessment in clinical practice. Each publication adds a new building block to the value proposition, and a sophistication of methods that help illustrate the value of the new drug compared to competing alternatives. The impact of OAB on patient HRQoL is explored through analyses of both disease specific and generic validated patient reported outcome (PRO) instruments. Utility values are also derived for the purposes of health economic modelling. The comparison of efficacy and safety of mirabegron with other competing alternatives is assessed using network meta-analysis (NMA), a requirement from most HTA bodies in the absence of head-to-head evidence. The outputs from the NMA and the PRO analyses are then applied to a series of trial based and NMA based HE models to assess the cost-effectiveness of mirabegron. This is followed by an assessment of effectiveness through analysis of a large retrospective database to see if indeed cost-effectiveness is supported in the clinical practice. The thesis concludes that cost-effectiveness of mirabegron is broadly supported by Real world evidence in terms of persistence and adherence while also highlighting the strengths and weaknesses of the current research and making recommendations for future research.

Introduction Diabetic kidney disease (DKD) is the leading cause of end-stage renal disease (ESRD), present in approximately 25%-40% of patients with long-standing diabetes and conferring additional risk of cardiovascular disease and mortality. Variations in the clinical presentations of DKD, heritability estimates from family-based studies and, more recently, the results from Genome-wide Association Studies (GWAS) demonstrate a heritable component of DKD. However, as is the case with the most of complex disorders, identifying causal genetic variants contributing to DKD has proven difficult. An important step in identifying variants associated with DKD in diabetes will involve integration of patient populations across multiple DKD cohorts, investigating rarer variants and by addressing the heterogeneity in DKD disease phenotypes in diabetes. Methods In this thesis, I reviewed the existing literature in genetic epidemiology in diabetic kidney disease. I then estimate chip-based heritability of DKD sub-phenotypes and replicated the association of known SNPS associated with renal function and upstream risk factors for diabetic kidney disease (BP, HbA1c) in patients with Type 2 Diabetes. I performed first GWAS for soluble receptor for advanced glycation products (sRAGE) a biomarker implicated in the pathogenesis of DKD. Finally, I performed GWAS for various DKD phenotypes on Type 1 Diabetes cohort (EURODIAB) and Type 2 Diabetes cohort (Go-DARTS) and helped with joint metaanalysis with DKD cohorts in SUMMIT consortium investigating genetic determinants of DKD. Results First, I showed that some DKD sub-phenotypes (like macro-albuminuria and ESRD) might be more heritable than others are and demonstrate that usefulness of estimation of chip-based heritability for complex trait by GCTA can be limited in the absence of large sample sizes. Second, I investigated the known genes for renal function (eGFR) and upstream risk factors for diabetic kidney disease (BP, HbA1c) in patients with Type 2 diabetes and showed that cumulative genetic risk for BP and HbA1c is associated with DKD. Third, I replicated the association of known loci associated with eGFR (UMOD GCKR and SHROOM3) in patients with Type 2 diabetes and showed that albuminuria affects the association of these variants with renal function. Fourth, I conducted a GWAS for sRAGE, an important biomarker associated with DKD, and identified novel variants in ITGA1 and HLA-DQA1 associated with circulating sRAGE levels. Finally, I performed GWAS for various DKD sub-phenotypes, and assisted in GWAS meta-analysis with SUMMIT consortium and identified potential novel genetic determinants for diabetic kidney diseases. Conclusion In conclusion this thesis has shown that a) estimation of chip based heritability of various DKD sub-phenotypes using GCTA has limited utility and requires GWAS studies with extremely large sample sizes b) the genetic determinants of renal function (eGFR) can interact with albuminuria in patients with T2D c) there are yet unidentified genetic markers associated with DKD and have identified potentially novel genetic markers associated with sRAGE (an important biomarker for DKD) and DKD itself which can be investigated in future studies for their reproducibility and functional consequences.

No description available.

Renal ‘progenitor’ cells expressing CD133 have been proposed as cellular therapeutics for treating patients with kidney disease. In the literature, CD133+ cells isolated from adult kidneys showed the expression of nephron progenitor markers (Pax2), and broad differentiation plasticity, being able to differentiate towards epithelial cells and podocytes. Most importantly, when injected into preclinical models of kidney injury, CD133+ cells integrated into damaged renal tissue and improved renal health. Nevertheless, the evidence for CD133 being a bona fide renal progenitor marker is conflicting. In this study, five renal biopsies belonging to children (from 6 months to 10 years old) were used. The localization of CD133 was consistent with previous studies, as the expression of CD133 was demonstrated on the cells of the Bowman’s capsule and in scattered tubular cells. From each sample, a bulk renal population was isolated and was initially characterized for the presence of the CD133 marker. Once placed in culture, most of the renal cells started expressing CD133. A further phenotypical characterization revealed that the vast majority of the cells expressed epithelial (EpCam, E-Cadherin, CD24), and some mesenchymal (CD73, CD44) markers. Also, the CD133+ population appeared heterogeneous for the expression of other markers. Most notably, CD13, a marker of fully differentiated tubular cells, was found to be significantly expressed in part of the CD133+ cells, suggesting that either fully differentiated cells started expressing CD13 de novo, or the CD133+ were committed towards a tubular fate. The bulk population was sorted by FACS into CD133+ and CD133- sub-populations which were compared in additional experiments to explore the progenitor-like features of the CD133+ cells. First, the ability of both CD133+ and CD133- sub-populations to differentiate towards podocytes in vitro was investigated. Both sub-populations were found to express the podocyte markers, nephrin and podocin, to a similar extent following stimulation with retinoic acid. However, the assay did not prove to be consistent and it was not used any further. Secondly, the potential of both CD133+ and CD133- sub-populations to integrate into ex vivo reaggregated mouse kidney rudiments was determined. Surprisingly, the majority of both cell types died in the chimeric rudiments within two days in culture. Neither the surviving CD133+ nor the CD133- cells showed any propensity to integrate into developing renal structures. Unexpectedly, the CD133+ cells were found to clump on top of the rudiment and had a negative impact on the developing rudiment, whereas the CD133- cells did not. Alongside with the test of the human cells in the chimeric rudiments, the assay itself was modified to suit the imaging of the rudiments in a Light-sheet fluorescent microscope (LSFM). 3D embryonic renal organoids were efficiently produced and the development of their structures could be successfully monitored through the LSFM in proof-of-concept experiments. The final aim of this work was to assess the therapeutic efficacy of the CD133+ and CD133- sub-populations in a rat model of cisplatin-induced acute kidney injury. The renal function was monitored using a non-invasive transcutaneous device that measures the half-life of an exogenously administered renal marker, FITC-Sinistrin, alongside to the measurement of conventional biomarkers, serum creatinine, and urea. Following intravenous (IV) injection, both CD133+ and CD133- cells ameliorated renal function and preserved renal histology. The data suggest that the human cells passed through the lungs, and probably reached the kidneys. However, no cells were alive at the end of the study (14 days), but traces of PKH26 were retrieved in lungs and, to a lesser extent, in the kidneys, suggesting the possible involvement of paracrine mechanisms, possible through extracellular vesicles in the observed functional amelioration. Additional biodistribution studies showed that soon after the IV injection the human cells were identified in the lungs of the animals, but not in the kidneys. Phagocytic cells, identified through the marker CD68, were observed around the human cells in the lungs as early as 1 hour after the injection. By 24 hours, clusters of CD68+ cells could be found, but not human cells. Therefore, the data suggest that the human cells die in the lungs and that the macrophages might play an active role in the disappearance. Taken together, this work shows that the expression of CD133 does not confer any advantage to the nephrogenic potential ex vivo or to the therapeutic efficacy in vivo. Moreover, since the cells were shown to be entrapped in the lungs, the renal repair is probably mediated by cell-derived factors, rather than by CD133+ cells homing to the kidneys and generating specialised renal cells. The role of macrophages in the resolution or regenerative mechanisms should be considered and further examined in future preclinical studies of cellular therapies for kidney diseases.

Benign prostatic hypertrophy (BPH) is a very common disease among men aged 50 and its economic burden on health services continues to grow. The advocation for adopting new screening procedure for BPH begins to emerge. However, a new proposal for screening should be under careful scrutiny and ineffective and inappropriate screening must be avoided. A prospective cohort study has been launched to study the frequency, distribution, and natural history of BPH in two well-defined small communities in Central Scotland. Using the data from the cohort study, the hypothesis that a BPH screening programme justifies the stringent criteria set by Wilson and Jungner (1968) to evaluate any proposed programme, could be tested. The hypothesis has to be rejected after taking all the criteria into account. BPH was a major health problem among apparently well middle-age and elderly men in the community. It imposed significant interference in men's daily routine as well as influenced on their psychological general well-being. Although there was a detectable asymptomatic stage, the natural history of BPH from asymptomatic to a clinical stage was not clear. Because of the obscurity of the natural history, the optimum interval between repeated screens of a continuous screening process was unknown. The facilities for diagnosis and treatment could not be met by the present health services. The economic implications of a screening programme could be enormous, though a systemic analysis to evaluate the worthwhileness of the screening programme in economic terms was not conducted.

No description available.

No description available.