Uraemia in end-stage renal disease patients leads to wasting of lean tissue, partly through the effects of acidosis that induce negative protein balance. Insulin resistance in these patients is also a major cause of muscle wasting, suggesting that low pH has a significant effect on insulin signalling in uraemic muscle. The pH sensitive SNAT2 amino acid transporter has been implicated in this because it is strongly inhibited by low pH, and amino acids are a well-established stimulus for the key protein kinase mTOR which regulates protein synthesis. The aims of this study were to determine: (a) the effects of amino acids, (especially L-Gln), and acidosis on insulin signalling and global protein synthesis/proteolysis rates; (b) whether these effects are mimicked by selective inhibition of SNAT2, and (c) whether intracellular amino acid depletion is sufficient to account for the functional effects of SNAT2 inhibition. In the L6 skeletal muscle cell-line, inhibition of SNAT2 with the nonmetabolisable SLC38 substrate methylaminoisobutyrate, metabolic acidosis (pH 7.1), or silencing of SNAT2 expression with smallinterfering RNAs, all decreased intracellular amino acid concentrations, mTOR activation, and global protein synthesis; and increased global proteolysis. Acidosis and small-interfering RNA inhibition both decreased phosphatidylinositol-3-kinase and protein kinase B activation, even though this is not regarded as an amino acid sensitive pathway. Extracellular amino acid depletion yielded decreases in intracellular amino acid levels similar to those observed during SNAT2 inhibition, but this failed to mimic the impairment of mTOR signalling observed when SNAT2 was inhibited. It is concluded that, in this muscle model, SNAT2 is able to regulate mTOR activation and protein synthesis rates; and that SNAT2 links acidosis, activity of the phosphatidylinositol-3-kinase/PKB signalling pathway and proteolysis, suggesting that SNAT2 is a key player in the acid-induced insulin resistance which is a prime cause of cachexia in acidotic uraemic patients.

Patients known to the kidney care services before needing dialysis should not start dialysis urgently through a temporary vascular access catheter, as this is likely to lead to increased morbidity and mortality. The aim of this study was to understand why patients within the East Midlands Renal Network start dialysis urgently when they have been known to the kidney care services for four months or more, and to make some recommendations on how to prevent this. An observational case-note survey found that patients who had an urgent dialysis start were more ill than those who had undergone elective dialysis initiation. Almost all had been known to the service for at least a year. They were less likely to have been seen in a predialysis clinic or to have attended a predialysis education session and older age was a significant barrier to an elective start. A qualitative study of patients who had recently started on dialysis showed that many had not been adequately supported psychologically, nor had their educational needs relating to self care and dialysis addressed throughout the disease process. A qualitative study of healthcare professionals suggested that they perceive distinct roles for themselves and for patients, with those who don't conform being judged unfavourably. Both studies revealed that are difficulties with of the timing of referrals within the kidney care service. Case studies highlighted the difference in perceptions of care between patients and healthcare professionals, the highly individual nature of involvement in illness management, and some difficulties with intercultural perceptions. Decision making preference varied but overall there was a lack of shared decision making. This thesis shows the need for a shift in the relationship between healthcare professionals and patients towards a more patient-centred, personalised approach, with efforts made to tailor education and decision making styles to each patient.

Note from the author: The financial support of the University Hospitals of Leicester is gratefully acknowledged in funding my training fellowship.

In the work described in this thesis the expression of activation antigens on human glomerular mesangial cells was investigated by selecting and subcloning hybridomas which secrete putative antibodies towards activated human mesangial cells. The panel of twelve monoclonal antibodies, produced in this way, was characterised to define the target antigens, cytokine receptors or cell adhesion molecules expressed as a result of mesangial cell activation. The twelve monoclonal antibodies were tested against non - stimulated mesangial cells and those stimulated with the growth factors PDGF or TGFβ1. Stimulation of the mesangial cells with PDGF or TGFβ1 was associated with an increase in the activity of the antibodies from tolerised mice. Comparing this significant difference in reactivity by these antibodies against non - stimulated and stimulated mesangial cells indicated that the antibodies were directed towards antigens expressed by stimulated mesangial cells. These antigens may be up - regulated antigens weakly expressed on non - stimulated mesangial cells or activation antigens expressed <I>de novo</I>. The reactivity of antibodies from the mouse immunised with PDGF stimulated mesangial cells against non - stimulated and PDGF stimulated mesangial cells was similar, indicating that the antibodies were directed towards cell surface antigens that are expressed to a similar degree by both non - stimulated and stimulated mesangial cells, that is, native antigens. The pattern of reactivity of antibodies from the mouse immunised with TGFβ1 stimulated mesangial cells, against non - stimulated and TGFβ1 stimulated mesangial cells, were similar to the reactivity shown by the antibodies from the corresponding tolerised mouse, indicating that the technique of tolerisation did not further enhance the specificity of the antibodies towards activation antigens. Seven antibodies of the panel of twelve monoclonal antibodies successfully detected antigens in normal and disease kidney. A striking pattern of reactivity against normal and diseased kidney was observed with three antibodies.

Inherited abnormalities of the kidney are frequently observed and represent a significant cause of morbidity and mortality. The globally increasing number of patients with end- stage renal disease (ESRD) urges the identification of molecular pathways involved in renal pathophysiology, to serve as targets for therapeutic intervention. Data from 2010 estimates the Saudi Arabian population to be 27 million, with one of the highest growth rates in the world. The population is characterized with high consanguinity rate, large family size, and a tribal structure. The consanguinity rate results in a high incidence of autosomal recessive genetic disorders. The population is at high risk of renal failure, with 133 incident cases per million populations per year that require renal replacement therapy. In such a population, characterization of new kidney disease gene loci using homozygosity mapping and positional cloning within consanguineous families is a powerful strategy. This study aimed to adopt this approach in order to search for known and novel molecular causes of inherited kidney diseases in the Saudi population. We studied patients and families with nephrotic syndrome, renal ciliopathies, nephrocalcinosis and renal agenesis. For nephrotic syndrome, we found that the most common genetic cause was a homozygous mutation in the NPHS2 gene. Novel and reported mutations in known nephrosis genes were detected. In a family with Bardet Biedl Syndrome, we utilized zebrafish and renal epithelial cells to determine the functional significance of a novel BBS5 mutation. In another consanguineous family with an autosomal recessive syndrome of distal renal tubular acidosis, small kidneys, and nephrocalcinosis we identified a novel locus on chromosome 2. We also describe the molecular genetic investigation of families with bilateral renal agenesis. In conclusion, in the highly consanguineous Saudi population we have utilized a variety of genetic approaches to identify and characterize novel genetic variants causing inherited renal disease.

Cystic kidney diseases are a fascinating cluster of discrete conditions and an important, common cause of established renal failure. Both isolated and syndromic inherited cystic kidney diseases are known to be linked by their pathogenesis involving ciliary dysfunction. Interestingly to date, all mutated genes which have been related to cystic kidney disease, encode proteins which are located on cilia, the basal body or centrosomes and are required for ciliary function. To date, over 50 causal genes have been identified and are capable of causing additional disease phenotypes, such as neurological disorders and blindness, often of variable severity. Understanding this clinical heterogeneity may considerably guide appropriate genetic counselling and screening of patients for relevant complications. Zebrafish are a well-recognised animal model, their advantages of: transparency; conserved genome; representative kidney and rapid external development; make them useful for studying organogenesis in the context of disease. Furthermore the ability to perform combined gene knockdown in zebrafish, to study the effect of oliogenicity, which was proposed to influence clinical phenotypes in cystic kidney disease related ciliopathies, was of interest. Using zebrafish models, this work studied the impact of four key genes, independently and in combination: ahi1, cc2d2a, nphp6 and mks3 on the development of cystic kidney disease and ciliopathy phenotypes, to resemble the human diseases nephronophthisis (NPHP), Joubert syndrome (JBTS) and Meckel Gruber syndrome, (MKS). A frequent finding in zebrafish morphants was a reduction in the number of cilia, which was usually associated with abnormal development of left-right body patterning and cystic kidney disease. Additionally, combined gene knockdown of: nphp6 and cc2d2a; ahi1 and cc2d2a; ahi1 and nphp6 was associated with a synergistic increase in disease phenotypes, suggesting an interaction between these genes. In conclusion, zebrafish are a powerful developmental model to study and ideally improve understanding of cystic kidney disease related ciliopathies.

No description available.

No description available.

In 436 children in whom urinary infection was suspected, bladder aspiration confirmed the diagnosis in 143 (26 male, 117 female). In a further 29 (9 male, 20 female) children, diagnosis was based on the demonstration of repeated significant bacteriuria and pyuria in midstream specimens of urine. Thus of the 436 patients who presented with suspected urinary infection, only 172 (40%) had proven infection. Of those patients (436) presenting with suspected urinary infection, only 24% under the age of 2½ years and only 50% over the age of 2½ years had proven infection. In the 172 children with urinary infection, there was a preponderance of females at all ages giving a sex difference between females and males of 4:1. One hundred and eleven (78%) of the 143 cases with urinary infection proven on suprapubic aspiration had a white cell count of more than 4 per cu.mm. in the bladder urine. In 106 children (42 male, 64 female) the urine obtained by bladder aspiration was sterile and in all but one of these cases the white cell count in the bladder urine was 4 or less per cu.mm. The clinical features of the 172 children with urinary infection at the time of presentation and of those with further recurrences during the course of follow up indicated that the symptoms of urinary infection under the age of 2½ years were non-specific i.e. fever, vomiting, diarrhoea, irritability, anorexia or feeding problem. In children over 2½ years, the main symptoms were dysuria, frequency of micturition, fever and abdominal pain. Enuresis was the main symptom in children with recurrent urinary tract infection. In respect of height and weight of children with urinary infection there was no difference whether they had normal or abnormal radiology of the urinary tract.

No description available.