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The different cellular stages of the spermatogenic cycle in man were identified and categorised from the array of cells observed in preparations obtained from whole tubules of the testis. This included description of meiotic figures. An attempt to establish the dynamics of the spermatogenic process was then made by two complementary approaches. Experimental work, initiated to establish and maintain differentiation in tissue culture, was successful. In vitro labelling with tritiated thymidine identified S phase spermatogonia and S phase primary spermato- cytes and followed the fate of these cells. At ten days, labelled primary spermatocytes were distinguishable from labelled spermatogonia and at 10-12 days a surge of unlabelled primary spermatocytes, identified at diakenesis, was observed. Changes in cell category frequencies were recorded with time in tissue culture and between individuals. Cell category frequencies were also studied in a series of infertile males. This revealed very considerable variation, with some categories not represented in some individuals. Significant relationships were found between some cell categories in the series. These trends allowed the cell categories to be assigned to the sequence of events within the spermatogenic cycle with some confidence. Reasons were given for assigning differences in cell category frequencies to a rate effect due to a combination of cell division events and the proportional duration of each cell stage. Previous models of spermatogenesis have concentrated on multiplication due to division to explain ratios between cell categories. One cell category was a particularly important factor for describing the activity of the germinal epithelium. A practical method of identifying points of lesion in the infertile spermatogenic cycle was described and compared with established methods for expressing the status of the infertile testis.

Neuroregeneration and repair of the spinal cord following traumatic injury (SCI) provide the ultimate goal in the treatment of this patient group and in the challenge to end the permanence of paralysis. To assess the effectiveness of these techniques as they are developed it is important to have sensitive functional assessment tools capable of detecting the repair of damaged pathways. Routine assessment of SCI subjects currently relies on subjective clinical measurement using the ASIA classification and Impairment Score, which is not always consistently rated and excludes autonomic function. In eight chapters this thesis describes work executed to fill this niche by, optimising and standardizing a somatovisceral neurophysiological tool that combines the evoked potential of the sacral somatic pudendo-anal reflex response (PAR) (as a surrogate marker of the pudendo-urethral reflex) with bladder function as a measure of the integrity of the guarding response (GR) for assessing residual supra-sacral bladder and sphincter function in SCI subjects. Presented is the confirmation of the predominant absence of the GR in complete SCI and its preservation, variability and inverse correlation with the degree of impairment in iSCI. Normally suppressed during voiding, the GR is enhanced in SCI, due to aberrant sacral reflexes leading to overactivity of the bladder and dyssynergia of the sphincters. For the first time shown is the residual control of the sphincter in SCI by facilitation of the PAR that gives the best systematic correlation with ASIA grading. And the localised cortical stimulation with TMS facilitates the PAR and may provide an additional means to test the precise integrity and timing of residual cerebro-spinal pathways facilitating the guarding response and pelvic floor muscles. The relationship of this somatovisceral measure with the ASIA/IMSOP scale has been confirmed and finally discussed is how the GR, through neurophysiological testing of somatovisceral reflexes could potentially present an assessment tool for those with SCI.

v. HA binding proteins (Hyaladherins) differentially affected extracellular HA structures. Inter-alpha-trypsin inhibitor (loci) is important in the formation of HA coats as well as HA cables. Tumour necrosis factor-stimulated gene-6 (TSG-6) is seen to be crucial to the formation of HA coats but not HA cables, while the role of versican in either structures is not fully determined yet although it is thought to be more crucial to the formation of HA cables.

Macrophages play a key role in renal inflammation and may be cytotoxic to resident cells within tissues. I begin this thesis by examining the effect of macrophages upon the level of apoptosis and proliferation in tubular epithelial cells <i>in vitro</i>. I then went on to examine the role of NO <i>in vivo</i> in the murine model of unilateral ureteric obstruction (UUO) characterised by tubular cell apoptosis and interstitial fibrosis. The specific iNOS inhibitor L-NIL (control D-NIL) was administered between days 5 to 7 following UUO. Mice were sacrificed at day 7 and the obstructed kidney removed for histological analysis. L-NIL treatment did not affect macrophage infiltration but did reduce both tubular and interstitial cell apoptosis. Proliferation of tubular cells and interstitial cells was unaffected. Interstitial fibrosis was significantly increased by L-NIL treatment. I also investigated the effect of conditional macrophage ablation in the UUO model. The conditional macrophage ablation mice used in these studies are transgenic for the human diphtheria toxin receptor (DTR) under the CD11b promoter (CD11b-DTR mice). Intraperitoneal (IP) administration of diphtheria toxin (DT) to DTR mice results in the rapid and specific depletion of monocytes and macrophages. DTR mice underwent UUO at day 0 and either DT or PBS was administered IP on days 5, 6 and 7. Mice were sacrificed at day 7 and the obstructed kidney removed for histological analysis. Administration of DT resulted in a 3-fold reduction in interstitial macrophage accumulation in obstructed kidneys. However, macrophage depletion had no effect upon proximal or distal tubular cell proliferation or apoptosis. Interestingly, macrophage depletion had no effect upon the accumulation of myofibroblasts but attenuated interstitial fibrosis.

Here we have demonstrated evidence of an angiogenic switch that occurs in human parathyroid tissue when diseased. Our data have not shown a significant expression of VEGF in these samples. A hallmark of chronic allograft nephropathy (CAN) is endothelial cell damage, resulting in hypoxia, providing a stimulus for angiogenesis, or new vessel formation. This study aims to compare vascularity of kidneys with CAN versus normal. In addition, validation of renal biopsy samples in the assessment of angiogenesis has been performed. CD31 staining from CAN nephrectomies exhibited diminished cortical and medullary staining and was accompanied by a significant increase in proliferation index when compared with normal. There was significant correlation in MVCs in core biopsies and the corresponding kidney cross-section. Early biopsies from grafts which developed CAN show significantly higher MVCs compared with the corresponding nephrectomy. The number of proliferating ECs was significantly increased in this biopsy group compared with normal kidney. Early protocol biopsies from grafts which developed CAN showed significantly higher MVCs compared with those with stable graft function (p=0.0002). This study demonstrates reduced density of CD31-positive microvessels in CAN compared with normal. In addition, the investigation of changes in the microvasculature in CAN by study of core biopsies has been validated and confirms preservation of normal vasculature in early CAN. Together, with evidence of proliferating endothelial cells these findings support a hypothesis that, early in the development of CAN, angiogenesis is stimulated, but that, despite this attempt at tissue repair, progressive microvascular loss results in interstitial fibrosis and eventual organ failure.

This thesis considers the hypothesis that small reductions in glomerular filtration rate (GFR), across the population range, are associated with a clustering of cardiovascular risk factors and increased risk of cardiovascular disease and death. The performance of a new method of assessing GFR, in which total plasma iohexol clearance is measured using dried capillary blood spots, was examined in a cross-sectional study of 81 consecutive individuals undergoing routine measurement of GFR. The new blood spot iohexol clearance (BSIC) method (using 3 blood spot samples) assessed GFR accurately compared to traditional iohexol clearance using 3 timed plasma samples; while prediction equations to estimate GFR from blood creatinine and cystatin c concentration performed poorly. Among 106 participants with measurements of BSIC-GFR, and GFR estimated from blood creatinine and cystatin c concentration, one or more measures of GFR were positively correlated with blood high density lipoprotein cholesterol concentration, and were inversely correlated with blood concentrations of triglyceride, C-reactive protein, fibrinogen, and homocysteine. BSIC-GFR was not more strongly related to cardiovascular risk factors than GFR estimated from blood creatinine concentration (eGFR). Cystatin c based GFR was strongly related to measures of body fat, while no relationship was seen with the other GFR measures. In a meta-analysis of cohort studies assessing the relationship between eGFR and risk of death and cardiovascular events, which included 4 061 003 and 1 372 820 individuals for each outcome respectively, a 30% lower eGFR was associated with a 20-30% increase in risk of both outcomes, depending on the type of study examined. however, there was significant heterogeneity between the studies. Large scale studies in which GFR is accurately measured are needed. Using the new BSIC method for this purpose is potentially feasible, but further work is required to ensure accuracy when the blood sampling is completed by participants themselves.

Mathematical modelling is utilised to provide potential explanations for the clearance characteristics of phosphate and beta2-microglobulin during chronic dialysis. The phosphate model is explored further with studies in acute renal failure and the effect of dialysis on intra-erythrocytic phosphate concentrations is assessed. Diurnal variation in phosphate concentration is explored. High haematocrit is not found to have a significant effect on the clearance of solutes across a wide range of molecule size. A four-pool model that can be applied in both acute and chronic renal failure is proposed to explain the observed kinetic behaviour of phosphate. Studies of intracellular phosphate concentrations fail to demonstrate release of phosphate from erythrocytic stores during dialysis. Diurnal variation in phosphate concentration is demonstrated in subjects with normal renal function and also in advanced chronic kidney disease. A multi-pool model explains the kinetic behaviour of beta2-microglobulin during dialysis. Beta-2-microglobulin deposition is assumed to be a staged process with some deposits easily accessible during dialysis and some more resistant to depuration. Patients receiving high-flux dialysis or haemodiafiltration are shown to have lower circulating beta2-microglobulin levels and less symptomatic dialysis related amyloid, but evidence of amyloid deposition is still found when assessed by a scintigraphic imaging technique. Age and duration of dialysis are shown to be the best predictors of symptomatic amyloid deposition. The results of the studies in this thesis indicate that, for solutes such as phosphate and beta2-microglobulin which have complex intra-dialytic kinetics, current dialysis techniques are insufficient to achieve adequate solute removal. It will be necessary to deliver longer or perhaps more frequent dialysis therapy in order to achieve this goal. Mathematical modelling facilitates understanding of the pathophysiology of the dialysis process and provides a platform for the development and monitoring of improved dialysis strategies.

Membranoproliferative glomerulonephritis (MPGN) and C3 glomerulopathy (C3G) are rare diseases that associate with dysregulation of the alternative pathway (AP). The earliest abnormalities associated with these diseases were C3 nephritic factor and also rare genetic variants in the gene CFH that caused factor H (FH) deficiency. Since then, other acquired and genetic abnormalities in AP have been reported in MPGN and C3G. The aim of this project was to screen cohorts of MPGN and C3G for such abnormalities. Screening for rare sequence variants in genes encoding proteins involved in AP activity in two cohorts revealed a low prevalence of genetic abnormalities. Compared to the prevalence of C3 nephritic factor and autoantibodies to complement proteins, it was clear that the predominant abnormalities in these cohorts were acquired. Though few, rare genetic variants identified in CFH were studied in functional studies. The first was identified in a case of familial MPGN in the N-terminal domain of CFH. Functional studies included surface plasmon resonance and haemolytic assays to study a mutant protein in the setting of a short fragment comprising the N-terminal domain of FH. This initial study confirmed loss of function in a familial variant and formed the basis for further studies. In studies of eight other variants identified in MPGN and C3G and two other diseases that share complement risk factors, only two variants were likely to be functionally significant as demonstrated by complete loss of function. This highlights the need for such studies to correctly identify important variants. The significant functional effects initially identified in studies using short fragments were then confirmed in studies using full length protein. The significance of rare genetic variants in CFH needs to be considered even though MPGN and C3G are largely an autoimmune phenomenon.