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Heparan sulphate (HS) plays crucial roles during the genesis and resolution of inflammation by sequestration, stabilization and presentation of proinflammatory cytokines and growth factors. The interaction between many of these factors and HS is critically dependent on the variable distribution of anionic 6-O-sulphated glucosamine residues within the structure of HS. The pattern of 6-O-sulphation is generated during HS biosynthesis by HS-6-O-sulphotransferases (HS6STs) but can be modified later by cell-surface HS-6-O-endosulphatases (SULFs). This study was designed to examine the potential contribution of these enzyme families to renal fibrosis following chronic inflammation. Initial experiments showed that the fibrogenic cytokine TGF-β induced SULF2 expression by renal tubular epithelial cells. Immortalized renal tubular epithelial cells were transfected to constitutively overexpress either HS6ST1 or SULF2 in order to examine the effect of these enzymes on cytokine function. Cells which overexpressed HS6ST1 showed increased binding of FGF2 compared to mock transfected control cells; this FGF2 binding correlated with increased pERK expression and enhanced cell proliferation. The requirement for HS for these processes was validated by inhibition of FGF2 binding with soluble HS, heparin or heparitinase III, whilst the importance of HS sulphation for increased binding was demonstrated after treatment of the cells with chlorate. Structural analysis of 35S-labelled HS from HS6ST1 overexpressing cells demonstrated an increase in mono-6-O-sulphated disaccharides accompanied by a decrease in 2-O-sulphated iduronic acid. By contrast, SULF2 transfectants showed reduced FGF2 binding, ERK activation and proliferation. Structural analysis of 35S-labelled HS from these cells showed a 50% reduction in 6-O-sulphation with a parallel increase in mono-2-O-sulfated iduronic acid. The significance of the in vitro study for renal fibrosis was then examined using a murine unilateral ureteric obstruction (UUO) model. Immunochemical analysis of UUO kidney sections showed a significant increase in expression of epitopes containing N- and 6-O sulphated HS around the renal tubules. This change was accompanied by a 5-fold increase in expression of the SULF1 gene. In summary, this study suggests that modulation of the expression of sulphate at the 6-O position in HS plays a significant role in the progression of chronic renal fibrosis by alteration of the biological activity of fibrogenic growth factors.

Haemolytic uraemic syndrome (HUS) is the leading cause of childhood acute kidney injury. It often occurs after a diarrhoeal illness caused by Shiga toxin (Stx) producing bacteria. HUS has significant morbidity and mortality but no direct treatment. The pathogenesis is incompletely understood. Initially considered an endothelial cell disease because glomerular thrombotic microangiopathy (TMA) is the pathological hallmark, this was challenged when patients treated with anti-vascular endothelial growth factor (VEGF) agents and mice with a podocyte specific Vegf knockout developed TMA and HUS. Affected children's kidneys and Stx treated cultured human podocytes also showed reduced VEGF. The study of familial atypical HUS also added to our understanding of the pathogenesis. It identified over-activation of the alternative complement pathway, leading to treatments blocking the complement cascade. Based on these data a hypothesis was generated. In Stx HUS, the podocyte is the initial toxin target. Stx binds to podocyte Gb3 receptors reducing VEGF secretion which decreases glomerular endothelial cell (GEnC) expression of protective complement regulators, making these cells vulnerable to complement mediated injury causing TMA and HUS. Human glomerular cells were tested for Gb3 expression and Std sensitivity. Both podocytes and GEnC expressed Gb3 but podocytes were significantly more Std sensitive. Consistent with previous reports, mouse podocytes did not express Gb3 and were Stx insensitive. Transfection with Gb3 synthase did induce Gb3 expression. An inducible, podocyte specific Gb3 synthase expressing mouse was made to study the central hypothesis. The podocytes of these mice expressed Gb3 but showed no glomerular abnormalities. They are now being challenged with Stx. The effect of VEGF on GEnC expression of complement regulators was studied. VEGF regulated GEnC expression of CD46 and CDS9 in a dose dependent manner. Serum based regulator factor H (CFH) was also synthesized and secreted and was up regulated by VEG F. Functional relevance of these changes was shown using a complement challenge assay. Significantly fewer complement deposits were seen on VEGF treated cells suggesting protection from complement attack. Kidneys from inducible podocyte specific Vegf knockout mice with TMA showed reduced glomerular CFH with evidence of complement deposition. In conclusion, human podocytes are sensitive Stx targets. Reduced VEGF secretion occurs after Stx challenge. loss of VEGF reduces GEne expression of protective complement regulators in a functionally significant way leading to complement deposition in a model of TMA. This work supports the central hypothesis. An inducible podocyte specific Gb3 expressing mouse has been created to test this further in vivo. This mouse is being studied to see if it develops HUS. If this occurs, it will allow the in vivo study of the pathogenesis of Stx HUS. Hopefully this will help identify potential therapeutic targets.

Acute kidney injury (AKl) and chronic kidney disease (CKD) are serious illnesses associated to high mortality and unsatisfactory therapeutic treatments. In search for new therapies, it has become evident that stem cells could be a possible option for patients with AKI and CKD. The evidence of the reno protective effect of bone marrow-mesenchymal stem cells (BM-MSCs) in experimental model of AKl, prompted us to study the effect of stem cells isolated from sources that are more accessible as cord blood (CB) and amniotic fluid. Infusion of hCB-MSCs in inununodeficient mice with AKI ameliorated renal function and tubular structure, prolonging survival. Moreover, transplanted hCB-MSCs localized in peritubular areas, limiting oxidative stress and apoptosis. By virtue of stem cell capacity to produce growth factors, hCB-MSCs were able- to induce the pro-survival factor Akt in tubular cells and subsequently their proliferation. Using the well-established model of AKI in immunodeficient mice, we studied the pro-regenerative effect of amniotic fluid stem (hAFS) cells. Infusion of hAPS cells in cisplatin-mice improved renal function and limited tubular damage, although not to control level, and prolonged animal survival. These cells engrafted injured kidney predominantly in peri tubular region and through a paracrine mechanism are able to exert an anti-apoptotic effect, to activate AId and stimulate proliferation of tubular cells. We enhanced the therapeutic potential of hAFS cells by cell pretreatment with GDNF, which markedly ameliorated renal function and tubular injury by increasing stem cell homing to the tubulointerstitial compartnent. In AKI models, the renoprotective effect of BM-MSCs is well established, however the role of these stem cells in model of eKD is controversial and not demonstrated so far. Therefore, we tested the effect of BM-MSCs in a model of adriarnycin-induced nephropathy. Repeated infusions of BMMSCs limited podocyte loss, and normalized distribution of parietal epithelial cells along the Bowman's capsule, reducing glomerulosclerosis. Moreover, through the local release of growth factors as VEGF, BM-MSCs were able to provide a local pro-survival environment that limited glomerular inflanunation and microvascular rarefaction.

The glomerular ultrafiltration barrier contains highly terminally differentiated podocytes with major processes and foot processes interlinked by ultrathin slit diaphragms. A number of molecules that are associated with nephrosis and podocyte damage have been described and these discoveries have given insight into the mechanisms that lead to podocyte injury. One of these molecules is CD2-associated protein (CD2AP), which is a crucial protein for slit- diaphragm assembly and function. In spite of the fact that CD2AP knockout causes nephrotic syndrome in mice and the heterozygous +/- mouse is prone to proteinuria, little is known about the relevance of this molecule in human renal pathology. In this thesis I studied the effect of a disease causing CD2AP mutation on the human podocyte phenotype. I identified the dramatic effects of the CD2AP mutation on the morphology of the cells and on the expression of mesenchymal, epithelial and other markers. I found that in contrast to wild type podocytes, CD2AP mutant podocytes acquired the characteristics of dedifferentiated cells. I compared the phenotypic characteristics of the CD2AP mutant podocytes with podocytes carrying mutations in the transcription factor WTI which is essential for normal nephrogenesis. Surprisingly, this study detected a similar de-differentiated phenotype in the CD2AP mutant podocytes to that seen in podocytes carrying a mutation in the WTl gene. In view of these phenotypic similarities I studied whether there is a functional link between CD2AP and WTI which confer these similarities in cellular phenotype. CD2AP is also known to bind to WTIP (WTl interacting protein), a molecule that binds WTI and can shuttle to the nucleus during podocyte injury. In this study, I found that WTIP might provide the functional link between CD2AP and WTl. In addition, in this study I have established the first conditionally immortalized human glomerular mesangial cell line with unique migratory properties, which will be an important adjunct in studies of representative glomerular cells, as well as in eo-culture studies. Overall this thesis demonstrates that there are clear implications for a novel role for CD2AP in the development and progression of glomerular disease. This thesis also discusses a novel conditionally immortalized human mesangial cell line that represents a new tool for the study of human mesangial cell biology in vitro.

Despite advances in its management, diabetic nephropathy continues to be the leading cause of end-stage kidney disease. Hyperplasia, hypertrophy, and extracellular matrix expansion are known to be present at the early stages of disease development, suggesting that a better understanding of the associated molecular mechanisms may point to novel therapeutic targets. The aim of this thesis was to investigate the mechanism and regulation of proliferation and cell growth in a cell culture model. In particular, the effects of metformin and rapamycin on the AMPKlmTORC1 signalling pathway were studied in mechanisms associated with the diabetic kidney. The results indicate that high ambient glucose differentially modulate the effects of metformin and rapamycin on mTORC1-related parameters related to proliferation and cell growth. These effects of high glucose have been associated with reduced AMPK activation. It has also been revealed that metformin and high glucose have an inverse relationship in the regulation of p21 CIP1N'JAF\ a cell cycle regulatory protein implicated in kidney hypertrophy, replicative senescence, and cancer. The results show that metformin inhibits proliferation and the expression of p21CIP1M1AFl in an AMPKa2-dependent manner. Metformin induced GO/G1 arrest and the associated down-regulation of p21 correlated with the level of cyclin D1, while rapamycin inhibited cell cycling without having an effect on these cell cycle regulators. These findings challenge the dogmatic view of p21clP1NVAF1 as a cell cycle inhibitor and may have relevance to the mechanism of high glucose-induced hyperplasia that precedes hypertrophic changes in the diabetic kidney. Several studies have linked increased GLUT1 expression to glomerular hypertrophy. However, despite increased cellular growth and extracellular matrix production, high glucose down-regulated GLUT1 expression in human mesangial cells in this study. Contrary to the general view; these findings suggest that in human mesangial cells a protective mechanism may operate against the harmful effects of hyperglycaemia.

Background and objectives: The majority of patients with chronic kidney disease (1) stage 3 are managed in non-specialist centres but most studies of CKD are conducted in Nephrology Departments in secondary care. Moreover, subjects ~75 years, which make up a large proportion, are often excluded. We undertook a detailed prospective study of unselected patients in primary care to fill the knowledge gap regarding CKD stage 3 and to identify factors that may be independent determinants of cardiovascular disease (CVO) and progressive CKD in this setting. Design, setting, participants and measurements: Subjects with eGFR 59- 30mUmin/1.73m2 were recruited from 32 Primary Care Practices. Medical history and demographic data were obtained and participants underwent clinical assessment including blood pressure, anthropomorphic measurements, skin autofluorescence (AF) and pulse wave velocity (PWV) as well as extensive urine and serum biochemistry. Results: 1741 participants were included: mean age 72.9±9 yrs; 60% (n=1052) female; 97.5% (n=1698) white; 17% (n=294) diabetic. Mean eGFR was 52.5±10mLs/min/1. 73m2 and 16.9% had microalbuminuria or more severe proteinuria. 77% had CKD stage 3A and 64.5% (n=1123) received RAASi treatment. 22.2% (n=387) had a history of cardiovascular events and 55% (n=950) were past or current smokers. Analysis identified participants with CKD stage 38, diabetes and age "275 years evidencing a higher risk profile for CKD progression and cardiovascular events than people with CKD stage 3A, those without diabetes or participants <75 years. 67% of participants required further medical intervention following their study feedback. Most required improved control of hypertension (n=576, 33 .1% of cohort). Less than 6% of participants met NICE criteria for referral to nephrology services. 41 % were unaware of their CKD diagnosis. Subjects with formal educational qualifications, age <75years, eGFR 30-44mUmin!1 .73m2 and significant albuminuria were identified as more likely to be aware of their diagnosis. Analysis of skin AF and PVVV readings revealed significant correlations with several potential risk factors for cardiovascular disease (CVO) and progression of CKD. Conclusion: The RRID study provides unique insights into patients with CKO stage 3 in primary care. Clinical characteristics are distinct from subjects generally seen in Nephrology Departments. Follow up over 10 years will assess the predictive value of a wide range of risk factors and yield valuable data complementary to that derived from studies conducted in secondary care.

Chronic kidney disease is common, affecting up to 25% of the elderly population. CKD is associated with both increased morbidity and mortality. The mainstay of treatment is blood pressure control. Whilst hypertension guidelines exist, there is significant concern amongst many practicing clinicians about their applicability to the older person. This concern is primarily due to falls risk. Falls are a significant problem in this age-group with a serious impact on those who fall, their families and the wider health economy. Whilst falls risk is multi-factoral, medication load (especially antihypertensives) is both significant and modifiable. The aim of this thesis is therefore to explore the risk-benefit relationship between blood pressure control, antihypertensive therapy and falls in older people with CKD. This was performed via an interlinked collection of clinical trials, culminating in an interventional drug trial. Traditionally such trials have focused on the effect of a drug (or combination of therapeutic agents), whereas we examined the effect of the therapeutic strategy of blood pressure control itself, as well as the effects of therapy beyond blood pressure itself. Our key results are: • Impairment of autonomic nervous system function can predict mortality in CKD, independent of other significant predictors. • Autonomic function is highly modifiable even in patients established on dialysis, and that these can be affected by factors such as PD fluid composition. • Body composition is highly variable over time in CKD. This is seen both in those on dialysis, and in pre-dialysis patients. • Antihypertensive therapy is associated with an acute reduction in circulating endotoxaemia. Whilst the long-term effect of this alteration is unknown, endotoxaemia is known to be associated with poor outcomes in many disease populations similar to CKD. • Antihypertensive therapy causes significant acute improvement in many markers of cardiovascular health in a cohort of older people with CKD (and non-CKD controls). Other effects of goal-directed AHT are also described, including changes to bone mineral biochemistry. • Antihypertensive therapy causes sustained improvement in many markers of cardiovascular health over a 12 month period in older people. Non-cardiovascular changes are also seen, some sustained from the original acute AHT use, whilst others regress towards baseline values. Falls remain relatively rare, and appear non-cardiovascular in origin. Cardiovascular disease in CKD is complex, and BP control remains a key component of its management. The data presented herein suggest that older people with CKD may benefit from aggressive risk reduction with AHT, just as young people do, and it is not appropriate to significantly modify this approach as a response to increased age alone.

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