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The role of reactive oxygen and nitrogen intermediates in the immune response of rainbow trout to R.s. was investigated. The early events occurring when the pathogen interacted with trout macrophages were assessed in terms of the respiratory burst elicited. Live R.s. elicited a respiratory burst, which was enhanced by heat-killed microorganism. This phenomenon, though, was not observed using UV-killed bacteria. Both responses were enhanced when a combination of LPS and TNF was used to activate the macrophages prior to contact R.s. Further studies demonstrated that both compounds synergised to enhance superoxide (O2) production, and that this was correlated with the ability to kill the pathogen. Opsonisation of R.s. with serum factors also increased the respiratory burst, but no difference was found between normal serum and heat-inactivated serum. The role of NO in the immune response of rainbow trout is also studied. Though no evidence of NO production was found in vitro, i.p. injection of live R.s. produced higher NO levels in serum as compared to controls. Fish injected with a virulent strain showed higher levels of NO than controls and than fish injected with an avirulent strain and other strains of unknown virulence. Fish vaccinated with killed R.s. and FIA also showed a significant increase in NO levels, but only four days after vaccination, decreasing thereafter, at both doses of vaccine tested. Injected of Brivax II, an attenuated strain of Aeromonas salmonicida, did not produce a significant increase of NO. RT-PCR was used to detect the expression of the iNOS in different tissues of rainbow trout. iNOS expression was seen only in gill and kidney after i.p. injection. iNOS was detected in the gills 6 h after injecting live R.s. and the expression was still present at day 5. iNOS was detected in the kidney 24 h after injection but was switched off at day 3. After bath challenge with the bacterium, iNOS was expressed in gill, gut and kidney, but the expression varied in each fish. No iNOS expression was found in macrophages isolated from challenged fish.

ABSTRACT This study was designed to screen antiretroviral therapy (ART)-naïve human immunodeficiency virus (HIV) infected patients for proteinuria, using urine dipsticks, at the HIV outpatient clinic at Johannesburg Hospital in an attempt to detect and treat early renal disease. In those with persistent proteinuria, a marker of kidney disease, renal biopsy was performed, ART with and without angiotensin-converting enzyme inhibitors (ACE-I) was initiated and patients were followed up for immunological and renal responses. After a minimum period of 12 months, a repeat biopsy was performed, where possible, to determine whether the histological lesions had responded to treatment. During urinary screening, proteinuria, leucocyturia and microscopic haematuria were common. Sterile leucocyturia may be associated with co-morbid sexually transmitted infection or tuberculosis. In the group that underwent renal biopsy with treatment, the renal and immunological response, before and after ART was highly statistically significant. Renal and immunological responses to ART were assessed by reduction in proteinuria with increased GFR, increased CD4 count with reduction in HIV viral load, respectively. On biopsy, HIV-associated immune complex disease was more common than HIVAN, a finding that contradicts international and some local data. Resolution of proteinuria was relatively rapid in comparison to the histological response to treatment, an effect not previously shown. This is the first study of its kind, to the author’s knowledge, that prospectively evaluates the effect of ART with/ ACE-I in ART-naïve HIV infected patients with both clinicopathological and histological criteria. It has shown unequivocally, that renal disease, particularly if detected and treated early in HIV infection, is responsive to treatment. These findings suggest screening for early detection and treatment of HIV-associated renal disease should be mandatory in HIV clinics in South Africa.

MSc (Med)(Genetic Counselling), Faculty of Health Sciences, University of the Witwatersrand / Autosomal recessive polycystic kidney disease (ARPKD; MIM263200) is a severe recessively inherited disease of the kidneys and biliary tract, with an incidence of approximately 1 in 20000 in non-isolated populations. It has a variable clinical spectrum from neonatal demise (in 30-50%) to survival into adulthood. ARPKD is caused by mutations at a single locus, polycystic kidney and hepatic disease 1 (PKHD1), with over 270 pathogenic mutations described to date. The high rate of compound heterozygosity in affected individuals has made genotype-phenotype correlations difficult. A common missense mutation, p.M627K, in exon 20 of PKHD1 was identified previously on the majority of ARPKD disease associated alleles in the Afrikaans population of South Africa suggesting the presence of a founder effect. The aim of this study was to describe the clinical phenotype of ARPKD in Afrikaans speaking individuals found to be homozygous for the common mutation, and to compare this phenotype to previously described cohorts of patients with ARPKD, known to harbour a spectrum of mutations. This descriptive study used retrospective data collected from records of patients with ARPKD at Johannesburg and Pretoria Academic Hospitals. Twenty seven individuals from 24 families were included in the study. Marked clinical variability was demonstrated within this subject group supporting the limitation of genotype-phenotype correlation described worldwide. ARPKD was diagnosed at a median age of 27 days, older than a North American cohort (NAC) born after 1990 (median age of 1 day). The majority (93%) of subjects in this study were diagnosed with chronic renal v insufficiency (CRI) and hypertension (HT), indicating the renal morbidities to be more common than noted in previous studies, but occurring at a later median age (1.4 years vs 13.5 days in the NAC). This may indicate a trend toward milder expression of renal morbidities in the present study. Portal hypertension was also diagnosed more frequently (81%) than in previous studies but at a younger median age (1.3 years vs 2.8 years), although with similar complication rates. Overall statistical correlation was found between the renal and hepatic related morbidities in this study, indicating that progression of the condition is not organ specific. A survival rate of 89% at one year is comparable to previous studies with similar patient ascertainment. This cohort represents the largest series of patients affected by ARPKD with a common mutation, described to date. The findings will provide for more accurate, specific and informative genetic counselling in families with ARPKD and may present a resource for future studies of modifier genes and environmental influences on the phenotypic expression of ARPKD.

Patients with chronic kidney disease (CKD) are a group with a markedly increased risk of adverse events following cardiac surgery. A particular problem for these patients is the development of post-operative acute kidney injury (AKI), which is associated with a significant increase in morbidity and mortality. Currently, there are no effective therapies proven to modify AKI in patients undergoing cardiac surgery. This thesis has three parts. The first describes an analysis of the Barts Health NHS Trust cardiac surgical dataset. Specifically, outcomes of patients with CKD and AKI were examined. The second describes a randomized control trial that examined the effect of remote ischaemic preconditioning (RIPC) upon AKI and myocardial injury in patients with CKD undergoing coronary artery bypass graft surgery (CABG). The final part describes the development of a panel of AKI biomarkers to allow the accurate prediction of AKI in patients with CKD undergoing CABG. The aims of this thesis were: 1. In our local cardiac surgical cohort, a. To assess the effects of CKD upon outcomes after CABG. b. To asses the prognostic importance of AKI after CABG. 2. To assess the potential for RIPC to reduce AKI and myocardial injury in patients with CKD undergoing CABG. 3. To investigate the diagnostic performance of serum and urine AKI biomarkers in a population of patients with CKD undergoing CABG. Analysis of the Barts Health NHS Trust cardiac surgical dataset confirmed that patients with CKD account for almost one-third of patients undergoing CABG. However, these patients account for a disproportionate two-thirds of all early mortality. CKD was also independently associated with late mortality after CABG. AKI was common in these patients. AKI was associated with late mortality even after accounting for pre-operative comorbidity and surgical complexity. In the randomized control trial, RIPC showed no effect upon the incidence of AKI or myocardial injury in the. 86 patients with CKD recruited. Secondary analysis of serum and urine biomarkers collected found change in serum cystatin C and NGAL as impressive predictors of AKI in patients with CKD. They allowed accurate early prediction of AKI more than 24 hours before diagnosis was possible using serum creatinine.

The increased risk of atherothrombosis in chronic kidney disease (CKD) has been under extensive examination for decades now. However, a treatment tailored for CKD patients is yet to be found. Current management plans can only tackle comorbidities and mostly fail. This thesis study examines the current literature related to CKD and thrombosis. The aim is to find a target suitable for therapeutic exploration. Normalizing the risk of thrombosis in CKD patients could curb a huge margin of their morbidity and mortality. In recent years, molecular biology studies attributed the extreme thrombogenicity in CKD to the retained uremic toxins. Indolic compounds are uremic toxins with a well described point of thrombotic activation. Of them, indoxyl sulfate is to be highlighted since it was shown to that it is one of the strongest pro-thrombotic uremic toxin. It is possible to therapeutically target this CKD specific cause of hyperthrombogenicity. Further research is very much needed in this area.

ASSOCIATION OF AGE, GENDER AND RACE IN CHRONIC KIDNEY DISEASE PATIENTS WITH AND WITHOUT DIALYSIS 1Busayo Adeyemi Onatolu, 2Hemang Panchal, 3Edward Francis Leinaar, 1*Shimin Zheng, 2Timir K. Paul 1Department of Biostatistics and Epidemiology, College of Public Health, ETSU, Johnson City, TN 37614 2Department of Internal Medicine, Quillen College of Medicine, ETSU, Johnson City, TN, 37614 3Department of Health Services Management and Policy, CPH, ETSU, Johnson City, TN 37614 *Sponsoring faculty Introduction: Studies have shown that chronic kidney disease (CKD) is common among adults in the United States. The Centers for Disease Control and Prevention (CDC) states that 30 million people, or 15% of US adults, are estimated to have CKD. Forty-eight percent of those with severely reduced kidney function are not aware of having CKD, and therefore do not receive hemodialysis (HD). Methods: A nationwide inpatient sample database from 2012-2014 was used to identify all patients admitted to the hospital using International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) codes (n= 534,845). Patients with dialysis dependent CKD (n=8,100) and CKD without dialysis (n=51,285) were compared to non-CKD patients (n=475,460). Hierarchical logistic regression was performed and p Results: Of the 534,845 patients, 88.9% were without CKD and 9.59% had CKD without HD and 1.51% had CKD with HD. Among patients with CKD, 13.64% were on HD and 86.34% were non-HD patients. The result shows that a higher proportion of patients with CKD without HD in the ≥ 80 years age group (≥ 80 = 37.84%, 65-79 = 36.94%, 50-64 = 20.80%, 35-49 = 4.12% and 18-34 = 0.30%) and a higher proportion of patients with CKD with HD in the 65-79 years age (≥ 80 = 16.30%, 65-79 = 41.79%, 50-64 = 33.09%, 35-64 = 8.09% and 18-34 = 1.29%). The OR of age group 18-34 compared to ≥ 80 is 5.690, 95% CI: 4.202,7.705, OR 35-49 is 4.552, 95% CI: 4.552, 95% CI: 4.103, 5.050, OR of 50-64 is 3.693, 95% CI: 3.444, 3.961 and OR 65-79 is 2.626, 95% CI: 2.457, 2.807. Males had higher rates of CKD than females, without HD (Male= 63.12%, female= 36.88%, p Conclusion: From this study, males had higher rates of CKD with and without HD than females, the age group ≥ 80 years had higher proportion of CKD without HD and those between 65-79 years had higher number of CKD with HD. Whites had higher rates of CKD with and without HD than other races.

Doctor of Philosophy (PhD) / Healthcare services and health research aim to improve the physical and psychosocial well being of consumers, and to offer responsive services needed and valued by them. Research in chronic kidney disease (CKD) has predominantly focused on investigating biomedical aspects and evaluating technological or pharmacological treatment interventions to improve medical management. While research into assessing patients’ and caregivers’ quality of life, and symptom burden, is growing minimal attention has been given to gaining a broad and in-depth understanding about the experiences, psychosocial issues and needs of patients and their caregivers. These need to be considered when planning and delivering patient-centred care and health research across the whole trajectory of CKD. The studies that form the major part of this thesis explore the perspectives, needs and experiences of CKD patients and their caregivers, within a broad and multidimensional framework encompassing aspects of the nature of the health and illness experiences and consumer perspectives. In Chapter 2, to understand what is known about parental experiences of caring for a child with CKD, the relevant qualitative literature was systematically reviewed and synthesized. Three inter-related clusters were identified: intrapersonal, interpersonal and external experiences. In Chapter 3, to gain a more detailed and broader understanding of this topic, in-depth interviews were conducted with parents of 20 children with CKD and 4 major themes were identified: absorbing the clinical environment, medicalising parenting, disrupting family norms, and coping strategies and support structures. In Chapter 4, to assess the effectiveness of support interventions for caregivers of patients with CKD, a systematic review was conducted which identified only three eligible studies that assessed only the effect of educational material on caregiver knowledge, not other domains. In Chapter 5, to describe and compare the broad range and depth of experiences and perspectives from predialysis, dialysis and transplantation patients, data from patient focus groups were analysed. The 5 themes that emerged from this data were: personal meaning of CKD, managing and monitoring health, lifestyle consequences, family impact, and informal structures. In Chapter 6, the focus groups were also used to elicit research priorities and identify reasons that patients used to develop their research priorities. A patient focused research agenda was elicited for CKD and 5 reasons that patients used to develop their research priorities were identified: normalisation of life, altruism, economic efficiency, personal concerns and clinical outcomes. During the focus groups, participants repeatedly expressed frustration about the poor public profile, and lack of community-based information on CKD prevention. So in Chapter 7, to assess how Australian news media covered prevention and early detection of CKD, I analysed television and newspaper stories that referred to CKD prevention or early detection. Kidney disease in general, and particularly the prevention and early detection of CKD, received virtually no media attention. When mentioned, it was mainly in the context of transplantation and donor stories, and seldom prevention or early detection, which appears largely unnewsworthy in its current form. At best, CKD received peripheral mention as a secondary concern in diabetes and obesity news stories which focused on lifestyle solutions. In Chapter 8, to develop a checklist for explicit and comprehensive reporting of qualitative studies (in-depth interviews and focus groups), I performed a comprehensive search in relevant publications for existing checklists used to assess qualitative studies. Seventy-six items from 22 checklists were compiled into a comprehensive list. All items were grouped into three domains: 1) research team and reflexivity, 2) study design, and 3) data analysis and reporting. The overarching purpose of these studies was to gain a better understanding about the needs, experiences and perspectives of CKD patients and their caregivers. The findings describe the permanent, profound and pervasive impact of CKD on the lives of patients and caregivers across the whole illness trajectory. A more detailed and broader understanding about patient and caregiver perspectives, as presented in this thesis, can support a move towards advancing patient-centred healthcare and research in CKD.

Six different isoforms of Type IV collagen (colIVα1-6) have been identified. The individual isoforms of colIV are termed alpha chains and are translated from six different COLIV genes (COLIVA1-A6). Collagen Type IV gene products compose the structural framework of basement membranes. The glomerular basement membrane (GBM) is a specialized basement membrane involved in the ultrafiltration processes of the kidney. The colIVα1-α5 chains are expressed in the human GBM while the colIV α1-α6 chains are expressed in the canine GBM. Many inherited diseases of the kidney have been reported and mutations in genes regulating kidney function have been identified. Alport syndrome (AS) is the most common form of human hereditary nephritis (HN). AS is defined as an inherited progressive kidney disorder associated with sensoneural deafness and is characterized by extensive thickening and multilamminar splitting of the GBM when examined by electron microscopy. AS has both X-linked (XLAS) and autosomal (ARAS) modes of inheritance. Mutations in the COLIVA5 gene are responsible for XLAS. A form of HN with characteristic splitting of the GBM with X-linked inheritance has been described in Samoyed dogs. A specific mutation in the COLIVA5 gene has been identified in Samoyed dogs affected with HN. Mutations in the COLIVA3 and COLIVA4 genes are responsible for ARAS. A form of HN has been identified in English cocker spaniel dogs (ECS) that has been described as autosomal in inheritance and includes GBM abnormalities including extensive lammination characteristic of ARAS. Both ARAS and ECS-HN show loss of the colIVA3 and colIVA4 chains in the GBM when examined with monoclonal anitibodies. ECS-HN has been hypothesized to have the same molecular basis of disease as ARAS. As such, we have isolated and characterized canine COLIVA3 and COLIVA4 sequences from normal dogs and ECS dogs affected with HN and compared the coding regions of these candidate genes.

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.