Mammalian kidney development proceeds through a series of interactions among metanephric mesenchyme, ureteric bud, extracellular matrix, growth factors and various other signalling molecules. These complex, but well integrated, networks control cell proliferation, differentiation, migration and survival and thus orchestrate kidney development. More and more molecules in these networks have been identified since the past few years. Therefore, it is crucial to explore their functions and the mechanisms by which they work to fill the research gaps. DACTs have recently been reported as pathway-specific regulators in WNT signalling, known to be important in kidney development, but their expressions and functions in mammalian kidneys are yet to be elucidated. The aims of this thesis are to describe the expression patterns of these two new genes, Dact1 and Dact2, in mouse embryonic kidneys and to further investigate their functional roles by applying RNAi to cell culture-based models. The first goal of this thesis is to establish the temporospatial expression patterns of Dact1 and Dact2 in kidneys by conventional end-point RT-PCR, quantitative real time PCR and RNA in situ hybridisation. Based on the expression patterns and preliminary observations in cell cultures, I hypothesize that Dact1 regulates cell proliferation while Dact2 governs cell migration. Experiments including siRNA transfection, BrdU proliferation assay, generation of stable cell lines expressing Dact2 shRNA and wound assay, are designed to test these hypotheses and the results may offer implications of functional roles of both molecules in kidney development. The results obtained are as follows. • Dact1 and Dact2 show different temporal expression patterns in mouse kidneys. In adult kidneys, Dact1 is greatly downregulated while Dact2 is still expressed at a comparable level to that at E14.5. • Dact1 is initially expressed in metanephric mesenchyme and, as development proceeds, shows a characteristic pattern of renal stroma whilst Dact2 is exclusively expressed in ureteric buds throughout embryonic stages. • Dact1 and Dact2 expressions are regulated by known regulators of kidney development including retinoic acid and chlorate. • Silencing of Dact1 facilitates proliferation of embryonic cells. • Silencing of Dact2 hinders migration of renal collecting duct cells. Taken together, I have characterised temporospatial expression patterns of Dact1 and Dact2 in kidneys and provided evidences of functional roles of both novel molecules in cell cultures. Based on this thesis, further studies on Dact1 and Dact2 using either in vitro or in vivo mammalian kidney models will offer more insights into their functions and regulations in renal organogenesis.

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.

Ca2+/calmodulin-dependent protein kinase type II (CaMK-II) is a multifunctional serine/threonine kinase that is ubiquitously expressed throughout the lifespan of metazoans. Mammals encode four genes (α, β, γ, δ) that generate over forty splice-variants. CaMK-II is important in a myriad of functions, including ion channel regulation, cell-cycle progression, and long term potentiation. In adults, alterations in activation of CaMK-II induce cardiac arrhythmias and heart failure. Developmental roles for CaMK-II are not as well understood since mouse knockouts are embryonic lethal. Therefore the identification of other vertebrate CaMK-II genes will add to our understanding of development. Zebrafish encode seven catalytically active CaMK-II genes (α1, β1, β2, γ1, γ2, δ1, δ2) due to a genome wide duplication event that occurred approximately 250 million years ago. Although, only 20-30% of all duplicated genes were retained, 75% of CaMK-II duplicated genes are transcriptionally active, pointing to a critical role for this signaling protein. mRNA expression patterns demonstrate that CaMK-II is expressed in diverse tissues including retina, pectoral fins, somites, heart, and kidney. Suppression of each gene generates unique phenotypes that mirror the mRNA expression patterns. Of the seven genes, camk2b2 and camk2g1 have the highest maternal contribution in zebrafish, are expressed in mesodermally derived organs, and develop defects similar to human syndromes. In fact, suppression of camk2b2 mimics the phenotype observed in zebrafish mutants of tbx5, the gene mutated in patients with Holt-Oram Syndrome. Camk2g1 morphants also exhibit similar defects as suppression of pkd2, the gene mutated in patients with Autosomal Dominant Polycystic Kidney disease. These roles implicate CaMK-II as an integral protein in the development and maintenance of mesodermally derived tissues.

Current immunogenetic tests before organ transplantation include HLA typing and detection of HLA-specific antibodies. However, these tests do not provide information about the B cells participating in the humoral response against the transplanted organ. BAFF (B activating factor) plays an important role in the proliferation, maturation and differentiation of B cells. A soluble form of the cytokine arises after splicing the membrane form of BAFF. The soluble cytokine binds to three types of receptors - TACI, BCMA and BCMA. Some recent studies suggest that BAFF could serve as a marker or predictor of antibody-mediated (humoral) rejection in kidney transplant recipients. Our study consists of two parts. The first part is focused on the detection of soluble BAFF levels in patients after renal transplantation. The aim of our study was therefore to correlate levels of soluble BAFF cytokine in patients before and after transplantation with the clinical course and incidence of rejection after transplantation. The study included 92 kidney recipients. Humoral rejection was diagnosed on the basis of a positive finding of C4d deposits in peritubular capillaries (imunoflorescenční detection), and the presence of donor- specific antibodies. BAFF levels were determined using Xmap methodology by the Luminex method...

Halogenated alkenes are a group of commercially important chemicals. For example tetrafluoroethylene is the monomer used for the production of poly- tetrafluoroethylene, hexachloro-1:3-butadiene is a by-product from the manufacture of chlorinated solvents and perchloroethylene is widely used as a dry cleaning agent. Due to possible exposure to haloalkenes and the nephrotoxicity observed in animal studies, concern has been expressed for the potential of these compounds to cause toxicity to man. Animal studies have shown that these compounds undergo inter-organ metabolism and are bioactivated by enzymes of glutathione processing. The metabolites are delivered to the kidney where they cause proximal tubular cell necrosis. This site-specific toxicity is due to accumulation of the metabolites via specific transport mechanisms and bioactivation via the enzyme C-S lyase present in high amounts in the proximal tubules. The aim of this research was to investigate the mechanisms of toxicity of haloalkene S'-conjugates in vitro using cultures of rat and human proximal tubular cells. This study demonstrates that human proximal tubular cells are sensitive to haloalkene. -conjugate toxicity, particularly DC VC. Human exposuredata has shown that workers exposed to trichloroethylene (Bimer et al, 1993) and perchloroethylene (Mutti et al, 1992) excrete nephrotoxic metabolites and markers of renal damage respectively. In the light of these findings and the toxicity of DCVC in HPT cells, exposure to halogenated alkenes should be controlled and those exposed monitored.

The renin-angiotensin system (RAS) is a major physiological regulator of body fluid volume, electrolyte balance and blood pressure. The principal effector peptides for this system are the angiotensins, particularly angiotensin II (Ang II), whose biological cations are mediated by specific angiotensin receptors. The use of highly specific nonpeptide angiotensin antagonists has allowed identification and characterisation of two major Ang II receptor subtypes, designated AT1 and AT2. The aims of this thesis were to investigate AT1 and AT2 receptor expression at both mRNA and protein level in freshly isolated and primary cultures of rat proximal tubular (PT) cells, and to determine the effect of culture upon Ang II receptor subtype expression. The possible role of Ang II upon receptor expression was also investigated. This study demonstrated that only the AT1 receptor is expressed in freshly isolated rat PT cells, with no evidence for AT2 receptor expression. Although continuously present throughout the culture period, a significant decrease in AT1 receptor expression was observed with time. Conversely AT2 receptor expression was absent in freshly isolated cells but was observed after 24 hours in culture with expression then remaining stable throughout culture. Clearly Ang II receptor expression is not stable during culture. Primary cultures of rat PT cells exhibit a change in receptor expression similar to those observed in vivo following tissue damage and repair, with an increase in AT2 receptor expression possibly mediated by locally released Ang II. Initial studies however, involving incubation of rat PT cells with exogenous Ang II, have demonstrated no effect upon AT1 receptor mRNA expression.

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), Bioethics and Health Law, Faculty of Health Sciences, University of the Witwatersrand / The current exclusion criteria for accessing renal dialysis in South African public hospitals places great emphasis on the allocation of scarce resources. The case of Soobramoney at the Constitutional Court highlighted the ethical and legal implications of providing this scarce resource. Mr. Soobramoney was denied access to renal dialysis on the basis of scarce resources and he did not qualify for care due to not meeting the criteria set for renal care. The Soobramoney case was considered mainly on the basis of scarce allocation of resources and offering treatment on an emergency basis. It was argued by the appellant that the state had an obligation to provide him with the treatment in terms of s 27(3) read with s 11 of the Constitution (para 14). This report takes a different slant and looks at the quality of life argument for increasing access to renal dialysis for those denied it based on current South African protocols. In exploring this concept one would venture to offer a definition of ‘quality of life’ according to Brown as an overall sense of well-being. This includes an individual’s satisfaction with their own lives (Brown, 2007: 72). A health related quality of life extends the definition to include the way a person’s v health affects their ability to carry out normal social and physical activities (ibid). A case is made for increasing access by developing programmes to cater for those in need of enhancing their quality of life. This is what is being motivated for in cases similar to Soobramoney, especially those with comorbid disease. The quality of life argument is based on the fact that there are indications in literature that patients with end-stage renal disease rate their own quality of life to be as important as the quality of life of the general population. Furthermore, there is no indication that the elderly live more miserable lives when they are on dialysis. The idea of respect for persons is highlighted - respect for the autonomous choices patients make concerning how they live their lives and including respect for them towards the end of their lives. Finally, I reflect on some legal issues concerned with the Soobramoney versus the Minister of Health Kwa-Zulu Natal 1997.

INTRODUCTION Cardiovascular diseases (CVD) are more common in kidney transplant recipients (KTRs) than in the general population. The high incidence of CVD in the KTRs can be attributed to traditional risk factors, additional risk factors associated with graft dysfunction and those specifically related to transplantation. Carotid intima-media thickness (cIMT) is a proven surrogate of atherosclerosis; it correlates with vessel pathology and is precisely imaged using ultrasound technology. This study was aimed at determining the prevalence and predictors of cardiovascular risk among KTRs at the Charlotte Maxeke Johannesburg Academic Hospital (CMJAH) and to examine the relationship between cardiovascular risk factors and carotid intima media thickness. METHODS Patients aged 18 years and above who received a kidney transplant at the CMJAH between January 2005 and December 2009 were recruited. A questionnaire that captured cardiovascular risk factors was administered. Patients records were assessed for information on their post transplant follow up. All patients had echocardiography and carotid doppler done for measurement of intima-media thickness. The Framingham Risk Score was used to categorize patients into low, moderate, high risk and very high risk groups. Results were analyzed using statistical package for social sciences (SPSS) version 17, p value of 0.05 was considered significant. RESULTS One hundred (KTRs) 63 male (63%) and 37 female (37%) were recruited ranging in age from 19 to 70 years, with a mean age of 42.2 ± 12.42. Thirty six patients (36%) were found to have high cardiovascular risk. Multiple regression showed proteinuria (p = 0.022), higher cumulative steroid dosage (p = 0.028), elevated serum triglycerides (p = 0.04) and the presence of plaques in the carotid artery (p = 0.012) as predictors of higher cardiovascular risk.Carotid intima-media thickness correlates with higher CVD risk. Fourteen patients (14%) had a carotid artery plaque. Twenty five patients (25%) had cIMT of >0.7 mm. CONCLUSION Kidney transplant recipients in CMJAH were found to have high cardiovascular risk (36%) and carotid intima-media thickness correlates with this high CVD risk. Routine follow up of KTRs should include measurement of cIMT as it provides a simple non-invasive assessment of subclinical atherosclerosis.

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.