Living kidney donation, the gift of a kidney from one individual to another for the purposes of transplantation, is a practice which goes against some of the most fundamental principles of medical ethics. Traditionally, it has been justified on the basis that the donor will benefit psychologically from donating, however data are limited that demonstrate this to be the case. The aim of this mixed-methods thesis was to explore the psychosocial aspects of living kidney donation, and to determine whether both positive and negative physical and psychosocial outcomes could be predicted. A qualitative study using in-depth interviews was conducted to identify the pertinent issues and to generate hypotheses. This included 23 pre- and post-operative living kidney donors. It was followed by a longitudinal quantitative study which utilised a number of both validated questionnaires and purposely designed questions to prospectively measure different psychosocial and transplant factors pre- and post-operatively. One-hundred living kidney donors participated. Additionally, a wound healing study was conducted to determine whether preoperative psychological factors had an effect on physical recovery. The surgical wounds of 58 living kidney donors were scanned using high-resolution ultrasound to see whether rates of wound healing were influenced by stress, personality and optimism. The final study explored unspecified kidney donation; a practice whereby individuals choose to donate a kidney to a stranger on the waiting list. As these donors do not know the recipient of their donation there is a concern that psychosocial outcomes in this group may differ to outcomes in specified donors (those donating to someone they know). A national consecutive sample of unspecified kidney donors was recruited to a retrospective questionnaire study. Questionnaire responses from 148 unspecified donors were compared with 148 specified donors; thus making it the world’s largest study of unspecified donation.

End stage kidney disease (ESKD) affects over 5300 people. The progression of chronic kidney disease (CKD) to ESKD is characterised by cytokine stimulation, leading to the activation of myofibroblasts, resulting in fibrosis. Kirsten rat sarcoma (KNRas) has a key role in the proliferation of renal fibroblasts in vitro and has been highlighted as a possible target for fibrosis. Previous research in our laboratory showed that inhibiting KNRas in the UUO model inhibited renal fibrosis. The aim of this thesis was to investigate the outcome of inhibiting KNRas using Antisense Oligonucleotide (ASO) in the novel Chronic Folic Acid Nephropathy (CFAN) model to study the effect on both fibrosis and renal function. The effect of inhibiting KNRas was also investigated in vitro, to try and elucidate the mechanism by which KNRas controls the progression of fibrosis. KNRas knockdown with ASO143 resulted in 50% reduction in KNRas mRNA which was associated with: 37%-50% reduction in total collagen and protection of renal function (BUN) in the 12 week CFAN model. TGFβ1 treated cells showed an upN regulation i KNRas, Jag1 and Collagen 1a mRNA. Treatment with KNRas ASO was associated with a 3.5 fold reduction in Jag 1 and a 55% reduction in collagen 1a. Jag 1 has been linked to the progression of renal fibrosis via biNdirectional signalling with Notch 1. In conclusion, ASO knockdown of KNRas inhibits fibrosis in vitro and in vivo, and in some instances protects renal function. These results support the hypothesis that KNRas targeting may be beneficial in the treatment of renal fibrosis. Further work is required to further understand the relationship between Jag 1,Notch 1 and KNRas, but this data suggests that KNRas affects renal fibrosis in a Jag 1 dependent pathway.

Diabetic Nephropathy (DN), a major cause of end stage renal failure, is believed to result from hyperglycaemia-induced pathways in the kidney. We previously identified μ-crystalline (CRYM) and Molybdenum Cofactor Sulfurase C-terminal domain-containing protein 2 (MOSC2) as two of several hyperglycaemia-induced renal genes in the Goto-Kakizaki (GK) rat, and showed that both were regulated by glucose in vitro in human mesangial cells (HMCs). In the current thesis, we investigated their expression in models of diabetes as well as in circulating cells from DN patients, in order to evaluate these genes as potential biomarkers and to explore their possible roles in the pathophysiology of DN. The expression of CRYM and MOSC were examined in tissues obtained from in vivo mouse models including a streptozotocin (STZ)-induced model of diabetes reversible through islet transplantation, renal cells cultured in normal (5mM) and high (25mM) glucose, and blood samples from diabetic patients with and without nephropathy (n= 97)/ retinopathy (n= 36). mRNA levels were determined using qPCR relative to reference genes, and protein location/abundance was determined using immunofluorescence. Functional analysis included N-hydroxylation assays. Both CRYM and MOSC2 mRNAs increased during hyperglycaemia in the diabetic kidneys of the STZ-induced mice and this increase was attenuated by treatment of diabetes (P < 0.05). Immunohistochemistry revealed an abundant expression of both proteins in tubular cells, and very low expression in mesangial cells. The hyperglycaemia-induced increase in renal CRYM was specific as we could not demonstrate any change in cardiac CRYM, and similarly no change in expression was found under diabetic conditions in vitro in Molybdenum Cofactor Sulfurase Cterminal domain-containing protein 1 (MOSC1), a homolog of MOSC2. Surprisingly, in high glucose (HG), CRYM and MOSC2 mRNA levels showed a slight decrease in cultured tubular cells, whereas they showed a significant increase in HMCs. Biochemical analysis of MOSC protein showed that hyperglycaemia increased the Nreductive activity of MOSC2 protein. To evaluate CRYM and MOSC mRNA levels as potential biomarkers of DN, we examined their expression in the peripheral blood of diabetic patients. We could detect low levels of CRYM but there was no expression of MOSC2. However, the closely related homolog MOSC1 was expressed in the peripheral blood though its mRNA levels did not change in association with DN. CRYM mRNA levels were 7.4-fold increased in patients with type 2 nephropathy and this effect was strongest in patients with a well controlled nephropathy compared to those with proteinuria. However, we found that circulating CRYM mRNA was reduced in patients with retinopathy. Therefore it is currently unclear if circulating CRYM is associated with nephropathy and its low levels of expression suggest it may not be a useful biomarker. Our data suggest that diabetes leads to an increased expression of renal CRYM and MOSC2 mRNAs, and although there are high levels of both mRNAs in tubular cells, the up-regulation may be taking place in mesangial or other renal cells. Circulating CRYM mRNA levels showed changes in patients with nephropathy and retinopathy but MOSC1 remained unchanged. CRYM or MOSC1/2 are unlikely to be useful biomarkers for DN but may be involved in the pathways that lead to DN.

Steroid Resistant Nephrotic Syndrome (SRNS) is characterised by either primary or late onset resistance to immunosuppression. Prognosis is poor and often results in rapid progression to end stage renal failure (ESRF). Generally, the incidence of disease-causing mutations in paediatric SRNS is 20-30%. 50 genes are currently associated with SRNS and since the advent of next generation sequencing, new genes are being linked each year. This study aimed to discover the true extent and spectrum of genetic variation in a comprehensive national NS population, selected by clinically relevant inclusion criteria. Patients were collected via a comprehensive national UK Renal Registry (RaDaR), which filtered recruits according to actual or presumed steroid resistance, and exclusion of secondary causes. Exome sequencing together with a stringent filtering algorithm was used to screen 187 paediatric SRNS and/or FSGS patients for the 50 genes associated with NS and to look for novel SRNS gene candidates. 25.7% of the sequenced patients had either, a previously described mutation or variant likely to be disease-causing. Detailed analysis revealed both known and novel disease-associated variations in the most common Nephrotic causing genes: NPH51, NPH52 and WTl and other more rarely associated genes such as LMX1B, MY01E, ADCK4 and the recently identified CRB2 and NUP107. Unexpected phenotypes were found in patients with mutations in genes usually associated with a different renal presentation, such as OCRL and DGKE. Genetic SRNS had no recurrence risk post-transplantation, whereas non-genetic SRNS had a 50% risk. Novel SRNS gene candidates e.g. MAGI2, along with potential risk factors were also found. This study demonstrates: 1. A broader genetic heterogeneity causing SRNS than previously realised and 2. A novel clinical and genetic stratification algorithm yielding a high rate of monogenic disease discovery and prognostic utility. Exome sequencing of this paediatric cohort also enabled novel SRNS genes discovery

Standard two-dimensional (20) ultrasound is widely utilised in diagnosis and intervention in urinary tract stone disease. 20 ultrasound as an interventional tool is limited to the well dilated renal pelvi-calyceal system. Three-dimensional (3~) and real-time (four-dimensional, 40) ultrasound has had minimal utility so far in the imaging of the renal pelvi-calyceal system and percutaneous intervention. The use of ultrasound technology also needs to be expanded to minimise radiation exposure to patients. The aim of this thesis is to explore the radiation exposure to patients in a stone episode and the application of three-dimensional ultrasound in the imaging of the renal pelvi-calyceal system (PCS). It also looks at the utility of 40 ultrasound for access in to less-dilated pelvi-calyceal systems, with the help of a dedicated model. Cumulative radiation exposure to patients undergoing stone investigation and treatment was assessed. We utilised an anthropomorphic renal model to perform measurements of the renal PCS using 20 and 3D ultrasound in order to validate 3D ultrasound use in the complex anatomy of the kidney. 40 ultrasound was investigated as a guidance tool in renal percutaneous intervention by building a dedicated interventional model and assessing accuracy of 40 ultrasound guided punctures with the help of fluoroscopy. Patients undergoing urinary tract stone diagnosis and treatment were exposed an average of 5.3 miliiSieverts (mSv) of radiation but could be as high 14.4 mSv. 30 ultrasound was found to be more accurate in assessing the renal PCS compared to 20 ultrasound. 40 ultrasound punctures were accurate and helped better placement of guidewires but overall accuracy was equivalent to 20 and some limitations were also seen. Radiation exposure to stone patients is a significant problem and imaging modalities like ultrasound need to be utilised more. 30 ultrasound proved accurate in the assessment of the complex anatomy of the PCS and showed promise as a guidance tool in intervention. However, some limitations were seen and technology advancements are awaited.

Sudden cardiac death accounts for one fourth of deaths in prevalent dialysis patients yet risk stratification for targeted preventive treatment remains an unmet clinical need. This thesis belongs to the field of non-invasive electrophysiology and its goal is to investigate if electrocardiographic monitoring during dialysis can provide subject specific profiles of selected indices of repolarisation and cardiac autonomic modulation derived with computerised analysis of the surface electrocardiogram that would be suitable for risk stratification purposes for sudden cardiac death in dialysis patients. To this end the following objectives were set: a) test the intrasubject reproducibility of selected repolarisation descriptors and of spectral parameters of heart rate variability (HRV) during 5 dialysis sessions, b) investigate their relationship with clinical and laboratory parameters associated with increased cardiovascular risk, c) examine the interrelationship between cardiac autonomic regulation and repolarisation indices, and d) follow up the study population for major arrhythmic events. The thesis is organised in a manner that allows selected chapters based on published articles to be read in their own right. A literature review on the epidemiology and possible underlying mechanisms of ventricular arrhythmias in chronic kidney disease is presented in Chapter 2. Chapter 3 deals with aspects of the study design and methodology. Chapter 4 includes the results on intrasubject stability of selected repolarisation descriptors. Chapter 5 reports on repolarisation descriptors in patients that suffered major arrhythmic events after a follow up period of 18 months. Chapter 6 reports on sex dependent association between HRV and pulse pressure and Chapter 7 reports an association between HRV and bone mineral abnormalities. T.he interrelationship between HRV and the studied repolarisation indices is investigated in Chapter 8. In Chapter 9 the future research plan is briefly outlined and Chapter 10 summarises and concludes the thesis.

Chronic kidney disease (CKD) is one of the major global health problems which may result in organ dysfunction. Podocytes are specialised cell type which plays a critical role in filtration. The interaction and attachment of podocytes to the Glomerular basement membrane (GBM) is critical for maintenance and functioning of filtration barrier. GBM is dynamic in nature and contains different proteins. Healthy maintenance of GBM is essential for filtration. Fibronectin (Fn) is identified as one of the components of the GBM. Fn is an established marker of fibrosis and involved in cellular functions such as adhesion, migration, growth, differentiation, signalling etc. EDA+Fn (Extra domain A) is documented as pathological alternative spliced variant of Fn. In our studies, we are studying the podocyte production, splicing and interaction of EDA+Fn in physiology and pathology. I have investigated the TGFpi mediated alternative splicing of EDA+Fn and underlying signalling mechanism in human podocytes culture and effect of EDA+Fn on podocytes phenotype and responses. Experiments were conducted on transformed human podocytes. Human recombinant TGFpi was used as a primary agonist. Fn spliced variants were measured by RT-PCR, Western blotting (WB) and Immunofluorescence (IF). Underlying intracellular signalling pathways were studied by using specific chemical inhibitors. The biological activity of EDA+Fn was assessed by TLR4 active HEK Blue cells. Effects of Fn spliced variants on podocyte phenotype and responses were studied by growing the podocytes on EDA+Fn and EDA-Fn coated dishes. TGFpi induces the production and alternative splicing of Fn in human podocytes which is regulated by PI3K/Akt and P38 MAP kinase signalling pathways. EDA+Fn was found to be biologically active as it activates the TLR4 pathway. EDA+Fn significantly alters the podocyte phenotype and responses. Targeting alternative splicing of Fn in human podocyte could be the influential remedial approach for the renal diseases.

Chronic kidney disease (CKD) is defined as a progressive loss of renal function measured through a decline in the estimated glomerular filtration rate. This occurs over months to years and is a significant public health concern with both the incidence and prevalence rates increasing throughout the world. Initial data was obtained from an Infinium HumanMethylation 450K Bead Chip array which investigated a total of 485,577 CpG sites in 255 individuals with CKD and 152 controls with no evidence of renal disease. Laboratory techniques including Sanger and next generation sequencing technologies were employed to elucidate further information regarding the top-ranked genes and microRNAs. Investigations were carried out using a range of samples from individuals with different phenotypes. Blood-derived DNA samples from individuals were also compared to their cell-line transformed equivalent DNA samples to determine the level of agreement between the genetic variation identified within both sample groups. Additionally, a meta-analysis of SNPs was completed for one of the top-ranked genes identified from the methylation analysis. Analysis of the data gained from the methylation array identified several genes and microRNAs which were significantly associated with the CKD population investigated. Further examination of these top-ranked results supported the generated methylation data, led to the identification of several SNPs and determined the differences in expression level between cases and controls. One significant association was determined from the meta-analysis which assessed different populations and ethnicities. The approaches undertaken considered tissue type and employed cost-effective analyses for both discovery and validation in order to explore the biological relevance of the top-ranked genes and microRNAs. The novel findings of both genetic and epigenetic modifications which are significantly associated with CKD, help to contribute to our understanding of the biological mechanisms which contribute to this disease.

The primary aim of this thesis was to examine major aspects of paternal genomics (by sperm DNA damage) and sperm protein expression (by proteomics) and their possible impact on male reproductive potential. In order to achieve this aim a number of studies were undertaken. To date, no relationships have been reported between ICSI outcomes and sperm DNA quality. Here, a novel assay specific to double stranded DNA damage was employed to see if a relationship was present: 1. The relationship between intracytoplasmic sperm injection (ICSI) outcomes and sperm DNA : fragmentation (single and double stranded DNA breaks) by alkaline and neutral Comet assays. A further attempt to find a biomarker for ICSI failure was performed using the sperm proteome associated with sperm DNA damage: 2. A study of seminal parameters and sperm proteome and their association with sperm nuclear DNA fragmentation following ICSI treatment. To identify areas of vulnerability within the male reproductive tract, a knock out animal model was employed to characterise DNA damage throughout the epididymis: 3. The characterisation of sperm DNA quality in HR6B (Ubiquitin-conjugating DNA repair enzyme) male knockout mice It has been reported that damage to the sperm genome and proteome can occur during routine lab procedures: 4. Here the effects of cryopreservation on the sperm proteome were assessed using high-throughput differential proteomics. The application of the well studied sperm DNA assays; alkaline Comet and TUNEL were compared with the novel neutral Comet in two disease models: a male reproductive and non reproductive cancer: 5. The characterization of sperm DNA quality in men presenting with Cancer.

It remains challenging to accurately predict whether an individual arteriovenous fistula (AVF) will mature and be useable for haemodialysis vascular access. In part, this is due to the heterogeneity of end stage renal disease (ESRD) populations studied as these have varied in terms of age structure, background ethnicity and the relative prevalence of different renal disease aetiologies. The natural history of AVF maturation is also confounded by multiple co-morbid conditions, such as diabetes and peripheral vascular disease, which may be present in ESRD patients. This thesis outlines the current practices for preoperative clinical assessment of patients coming to AVF formation and explores the potential novel methods of predicting AVF patency outcomes. We characterise the age related mortality of patients undergoing AVF formation revealing a high post operative mortality for those over 75 years of age and present a meta-analysis of AVF outcomes in elderly patients demonstrating significantly poorer outcomes for distal versus proximal fistulas. We analyse the impact of surgical training on AVF outcomes concluding that results are not adversely affected by supervised surgical trainees. We also present the results of implementing the recommendations from international longitudinal studies to establish a ’Fistula Fast' pathway and examine the impact this has on AVF outcomes and its subsequent reduction in central venous catheter usage in our unit. Finally, this thesis presents preliminary work in diagnosing arterial stiffness and endothelial dysfunction using non-invasive methods of pulse wave velocity and near infra-red spectroscopy and, explore how these results can be used as a novel predictor of AVF outcomes in the ESRD population.