This thesis studied a rat model of acute and reversible ureteral obstruction in which there is no necrosis and in which renal functional abnormalities are transient. A leukocyte infiltrate was found to be one of the earliest responses of the kidney to ureteral obstruction, occurring within four hours of obstruction, with the peak response occurring at 12 hours. The leukocytes form distinctive rings around the tubules, particularly distal tubules. The leukocyte cell infiltrate consists predominantly of macrophages with a lesser number of T-lymphocytes mainly of the cytotoxic, suppressor cell subclass. The kinetics of the macrophage invasion temporally correlates with both the decline in glomerular filtration rate and enhanced thromboxane B2 excretion. In addition the infiltrate correlates with alterations in tubular function. Total body irradiation of rats prior to the onset of obstruction so as to prevent the leukocyte infiltrate in the kidney, both reduces thromboxane B2 excretion and significantly improves renal haemodynamics after release of 24 hours of obstruction. This implies that infiltrating leukocytes contribute to the decline in glomerular filtration rate and renal plasma flow seen after obstruction, possibly via the production of vasoactive prostanoids such as thromboxane A2. The elimination of the leukocyte infiltrate from the obstructed kidney, however, does not return the function of the post-obstructed kidney to normal. Glomeruli isolated from rats with ureteral obstruction demonstrate an enhanced ability to produce a variety of prostanoids, including thromboxane A2 and this also appears to play a role in the functional changes following ureteral obstruction. The cortex of the obstructed kidney produces a unique chemoattractant that is specific for macrophages. This substance behaves biochemically as a lipid, and may account (in part) for the macrophage infiltrate following ureteral obstruction.

Detrusor instability is the commonest type of urinary incontinence in the elderly and is present in up to 50% of patients attending continence clinics. Treatment of this condition, aimed at reducing uncontrollable detrusor contractions, is at present unsatisfactory. For example, calcium antagonists are cliniclly disappointing and studies were carried out to investigate why they are ineffective. Rats were treated with nimodipine for 8 days or with a single dose. Treatment for 8 days had no effect on isolated detrasor contraction but a single dose reduced detrasor contractile response. It is propossed that chronic treatment with nimodipine caused an up-regulation of calcium channels as a compensatory mechanism. Oestrogens have been shown to have an inhibitory effect on detrusor muscle contraction after in vitro and in vivo treatment. In post-menopausal women with a uterus unopposed oestrogens should not be given, but progesterone has anti-oestrogenic actions. When rats were treated with oestrogen and progesterone for 8 days, there was no effect on rat detrasor contractile response. An anti-oestrogenic effect of progesterone has therefore been demonstrated in rat detrusor smooth muscle. Caffeine has been shown to increase detrasor pressme on bladder filling in patients with detrusor instability. The effect of low concentrations of caffeine on the contractile response of isolated human and rat detrusor muscle was therefore determined. Caffeine was found to have only a slight potentiating effect on isolated human and rat detruosr muscle contraction. The results in this thesis have important clinical imphcations for the treatment of detrusor instability. It may be more effective to administer calcium antagonists in an intermittent manner. Oestrogens are better given alone or with the lowest possible dose of progestogens. Caffeine would not be contraindicated in patients with detrusor instability.

No description available.

In vitro expanded neural stem cells provide an important cellular model to explore mechanisms of neural development, for modelling of disease, and in the longer term may have applications in new types of stem cell-based therapies. However, our ability to steer neural stem (NS) cell lines into specific desired lineages in vitro remains limited. PDGFRα is one of the earliest markers of the transition of neural stem cells to oligodendrocyte progenitors. I established and characterised a novel set of mouse NS cell lines that report the activation of PDGFRα via expression of an H2B:GFP ‘knock-in’. Three clonal ‘PG1’ cell lines were fully characterised. Under self-renewing conditions I found <1% of NS cell express the H2B:GFP reporter but this increases to ~15-20% following induction of differentiation. Using this cellular model system I carried out a high-content chemical screen of a diverse collection of 463 pharmacologically active small molecule modulators of ‘stem cell pathways’ and kinase inhibitors, to identify those capable of modulating NS cell self-renewal and differentiation. I did not uncover any small molecules capable of promoting OPC lineage specification. However, I found multiple HDAC inhibitors that were highly effective in blocking the activation of PDGFRα, a finding that mirrors published studies implicating HDAC inhibition in the later differentiation of OPCs to oligodendrocytes. Three further compounds, Nigericin (an ionophore), Withaferin (a steroidal lactone) and NFkB inhibitor also completely blocked OPC commitment. I focused on the specific cellular responses and downstream molecular events triggered by these molecules and tested their differentiation potential on human NS cells and malignant glioblastoma-derived NS cells.

Chronic Kidney Disease (CKD) is a risk factor for contrast induced nephropathy (CIN), defined as an increase in serum creatinine of >25% from baseline or a delta rise of >26.5% μmol/L within 48 hours. This thesis describes the assessment of novel biomarkers for the earlier prediction of CIN. A prospective observational study of 301 CKD patients was performed. Demographics and a Mehran risk score were recorded. Low-osmolar contrast was used. Samples for plasma and urinary neutrophil gelatinase-associated lipocalin (NGAL), serum liver fatty acid-binding protein (L-FABP), serum kidney injury marker 1 (KIM-1), urinary cystatin C (CysC) and serum interleukin 18 (IL-18) were taken at 0, 1, 2, 4, 6 and 48 hours post contrast. Major adverse clinical events (MACE) were recorded at 1 year which included acute myocardial infarction, heart failure, stroke and death. CIN occurred in 28 (9.3%) patients and was associated with older age, diabetes, higher Mehran score, larger contrast volume and anaemia (p <0.05). NGAL performed best at 6 hours with levels of 1,337 ng/ml in AKI patients compared with 931 ng/ml in non-AKI patients, p=0.002, AUC 0.71, sensitivity 75.0%, specificity 96.1 %, OR 2.86. L-FABP performed best at 4 hours with levels of 10.7 ng/ml in AKI patients compared with 6.2 ng/ml in non-AKI patients, p=0.001, AUC 0.69, sensitivity 42.3%, specificity 90.2%, OR 6.75. Median urinary NGAL was higher only after 48 hours, 487 ng/ml in AKI patients versus 155 ng/ml in non-AKI patients, p=0.008, AUC 0.63. CysC, IL-18 and KIM-1 were not predictive at any time-point (p>0.05). A Mehran score >-10 achieved an AUC of 0.65, p=O.006. MACE occurred in 7 (25%) AKI patients and 17 (6.2%) non-AKI patients (p<O.001) with a higher mortality in AKI patients (3 patients [10.7%]) compared with non-AKI patients (8 patients [3.3%]) respectively, p = 0.037. In conclusion, a higher Mehran risk score, 6 hour NGAL and 4 hour L-FABP performed best at earlier prediction of CIN. AKI patients were significantly more likely to develop MACE or have a higher mortality at one year.

Familial clustering of diabetic kidney disease (DKD) suggests the existence of a genetic predisposition towards the development of the disease. DKD continues to be the leading cause of end-stage renal disease (ESRD) worldwide. The identification of factors associated with a higher risk of DKD is an important scientific goal. Novel biomarkers associated with DKD may prove useful for the clinical prediction of DKD. At the beginning of this project the key research theme was to explore the genome-wide association study (GWAS) data generated by the GEnetics of Nephropathy, an International Effort (GENIE) consortium. The GWAS dataset was derived from -2.4 million single nucleotide polymorphisms (SNPs) genotyped in a large case-control collection with multi-centre replication (n>12,OOO individuals). This initial project was promising, stimulating several additional projects assessing both mitochondrial and telomere-related genes and their associations with DKD. Genetic variation in the form of SNPs does not explain all of the inherited component of DKD so DNA methylation, was considered using data from an epigenome-wide association study (EWAS). This too was conducted on mitochondrial and telomere-related genes. The final stage of research within this thesis was to comprehensively evaluate genetic polymorph isms within the mitochondrial genome using next generation technology employing the Ion Torrent Personal Genome Machine (PGM) and Illumina TruSeq Genome Analyser (GAll).

Diabetic nephropathy (ON) is a major microvascular complication associated with diabetes. DN is characterised by a progressive loss in renal function and associated renal fibrosis. Currently, no cure exists for DN and therapeutic strategies seek to slow the decline in renal function, through glycaemic and blood pressure control. Acute kidney injury (AKI) is a condition that can arise due to a range of pre-renal, renal and post- renal causes. AKI is associated with decreased glomerular filtration rate (GFR), resulting in accumulation of nitrogenous waste products and other complications. The precise molecular mechanisms of renal damage in these diseases is still not fully understood and needs to be further elucidated. Bone morphogenetic proteins (BMPs) have been shown to be anti-fibrotic in models of renal fibrosis. Gremlin1 (Grem1), a BMP antagonist is upregulated in conditions such as ON. It is hypothesised that Grem1 binding to BMPs is crucial to its pathogenic role in renal fibrosis. The specific interactions of Grem1 and BMPs in disease have not been fully determined. The overarching aim of this thesis was to better define the role of Grem1 binding to BMPs in vitro, in order to contribute to our understanding of these interactions in the context of kidney disease. Using a range of in vitro methods, we determined that Grem1 relative binding affinity is BMP-2>-4>-7, and that the post-translational modifications of glycosylation and phosphorylation of Grem1 may not be essential for BMP antagonism. We also found that Grem2 differentially inhibits BMP-2, -4 and -7 signalling in vitro. We determined that isolation of Grem1-BMP complexes are difficult using standard in vitro methods, and that direct mass spectrometry (MS) approaches may be advantageous. We have determined that mice with tubular specific deletion of Grem1 (Grem1 TEC -1-) display somewhat attenuated renal damage in a folic acid (FA) induced model of AKI. These findings provide a mechanistic insight into the possible molecular interactions between Grem1 and BMPs, and may have implications in the context of renal fibrosis. Given the high binding observed between Grem1 and BMP-2, this complex formation may be favoured in the 'diseased' kidney. Furthermore, Grem1 inhibition of BMP-7 may not occur via direct interaction. Manipulation of the Grem1-BMP axis has been explored as a potential therapeutic strategy by many research groups, and this is further supported by our findings that Grem1 TEC -1- mice display attenuated renal injury in a model of AKI. Further work will be required to further define these interactions in models in renal fibrosis.

Proteinuria has been known for about a century, yet much information concerning the renal handling of protein, particularly by the normal kidney, requires further and more thorough investigation. Since then, some theories have been put forward and assumptions have been made to explain the mechanism of renal function with respect to the glomerular filtration of endogenous or exogenous infused proteins. However, the ways by which these proteins are acted upon by the kidney are still incompletely understood. Although the renal tubular handling of plasma proteins by the diseased kidney has been extensively investigated, this handling is still in need of further study especially in the case of protein not normally present in the plasma, and in the normal kidney. Furthermore, there is some evidence that in proteinuria, the renal tubular reabsorption of plasma proteins is non- selective. Although such a phenomenon is probably operative in the case of plasma proteins, there is evidence that filtered proteins which are not normally present in the plasma may not be reabsorbed at all in the tubules, or only to a limited extent. The most important protein which has been extensively investigated in this respect is haemoglobin, but even attempts to demonstrate histologically that haemoglobin enters the renal tubular cells have yielded equivocal results. In consequence the author's aim was to investigate another important tissue protein and its handling by the normal mammalian kidney. Homologous erythrocyte carbonic anhydrase (C.A.) was chosen for this study for several reasons, the most important of which may be summarised as follows;- (a) C.A. may be detected in the urine of patients suffering from intravascular haemolysis and is absent from those with extravascular haemolysis. (b) It may be easily prepared in fairly good yield and in an immunologically pure state, as a single protein component of homogenous molecular size. (c) The low molecular weight of C.A. is well established (30,000), hence it may be easily filtered through the glomerulus. (d) This enzyme is present neither in normal blood plasma nor in normal urine. Therefore, it was thought that some light might be thrown on the renal tubular mechanism in the case of induced proteinuria, if the clearance of this protein by the normal mammalian kidney were investigated.

Hyperphosphataemia in patients with advanced chronic kidney disease (CKD) is thought to be an important contributor to cardiovascular risk, in part because of endothelial cell (EC) dysfunction induced by inorganic phosphate (Pi). Such patients also have an elevated circulating concentration of pro-coagulant endothelial microparticles (MPs), leading to a pro-thrombotic state, which may contribute to acute occlusive events. It is hypothesised that hyperphosphataemia leads to MP formation from ECs via an elevation of intracellular Pi, which directly inhibits phosphoprotein phosphatases, triggering a global increase in phosphorylation and cytoskeletal changes. Using cultured human endothelial cells (EAhy926), incubation with elevated extracellular Pi (2.5mM) led to a rise in intracellular Pi concentration within 90min. This was mediated by PiT-1/slc20a1 Pi transporters; and led to global accumulation of Tyr- and Ser-Thr phosphorylated proteins, a marked increase in cellular Tropomyosin-3, plasma membrane blebbing and release of 0.1 – 1 micron diameter MPs. The effect of Pi was independent of oxidative stress or apoptosis. Similarly, global inhibition of phosphoprotein phosphatases with orthovandate or fluoride also yielded a global protein phosphorylation response and rapid release of MPs. The Pi-induced MPs expressed VE-cadherin and superficial phosphatidylserine, and in a thrombin generation assay were significantly more pro-coagulant than particles derived from cells incubated in medium with a physiological level of Pi (1mM). These data demonstrate a mechanism of Pi-induced cellular stress and signalling which may be widely applicable in mammalian cells; and in ECs provides a novel pathological link between hyperphosphataemia, generation of MPs and thrombotic risk.

Despite the importance of extracellular phosphate, the mechanisms and control of intestinal phosphate transport remain unclear. The present study used in vivo and in vitro methods to compare the extent of Na+- dependent versus Na+-independent phosphate transport along the rat small intestine and colon at different luminal phosphate concentrations. Na+-dependent and Na+-independent phosphate transport and genomic expression of type II (NaPi-II) and type III (PiT) transporters in young (3- week old) and adult (8- and 16-week old) animals fed a control or low phosphate diet have also been quantified. mRNA levels of Na+- dependent phosphate transporters have been analysed in the 5/6 nephrectomy model of chronic renal failure and following treatment with matrix extracellular phosphoglycoprotein (MEPE). The acute effects of altered luminal phosphate concentration on intestinal phosphate transport and renal phosphate transporter expression was also assessed. The findings confirm the jejunum to be the main site of Na+-dependent phosphate absorption under both normal and low dietary phosphate conditions. Low phosphate diet upregulates Na+-dependent and Na+- independent phosphate transport in both duodenum and jejunum, whereas age only affects Na+-dependent component in the duodenum and jejunum. When luminal phosphate concentrations are in the physiological (millimolar) range, absorption in the jejunum displays less Na+-dependency and is unlikely to be mediated exclusively by the Na+- dependent NaPi-IIb cotransporter. In the ileum, again using millimolar luminal phosphate concentrations, significant Na+-dependent phosphate transport was detected, but the rate of phosphate absorption was lower than in the jejunum. Since NaPi-IIb is not expressed at the rat ileal brush-border membrane (BBM), the presence of significant Na+- dependent phosphate transport suggests an alternative phosphate uptake pathway in the ileum. At millimolar luminal phosphate concentrations in the distal colon, only Na+-independent phosphate absorption was detected. Thus, at concentrations of phosphate normally present in the intestinal lumen, Na+-independent pathways of phosphate absorption are present in the proximal small intestine and distal colon, as well as a non-NaPi-IIb-mediated Na+-dependent pathway in the distal small intestine. PiT2 assumes a dominant role in phosphate transport in the jejunum of pre-weaned animals, and in the kidneys of animals fed a low phosphate diet. PiT1 transporter is not regulated by age or low phosphate diet at the genomic level. Chronic renal failure reduces intestinal and renal expression of the major transporter NaPi-II, and also PiT1 and PiT2. However, MEPE reduces intestinal expression of NaPi-IIb and PiT2 alone in chronic renal failure animals. Acute duodenal instillation of either 15 mM or 1.3 M phosphate did not affect the renal BBM protein expression of NaPi-IIa and NaPi-IIc. Duodenal instillation of 1.3 M, but not 15 mM, phosphate increased phosphate uptake in the duodenum only; however, the small intestine is unlikely to encounter phosphate concentrations in the molar range.