No description available.

No description available.

High concentrations of nitric oxide (NO), produced by the inducible form of nitric oxide synthase, expressed by proximal tubular (PT) cells and invading macrophages, may damage the PT during inflammatory renal disease. The natriuretic peptide hormones (NPs) regulate inducible NO production in a variety of cell types.  The aim of our studies was to investigate the regulation of inducible NO synthesis in macrophages and PT cells by the NPs. Cultures of rat and human PT cells and murine macrophages cell lines were incubated with combinations of cytokines and/or lipopolysaccharide (LPS) (to stimulate NO synthesis), NPs and pharmacological agents.  Nitrite was measured as an indicator of NO production using the Griess assay.  To investigate more directly the possibility of regulation of NP production by NP mediated interactions between PT cells and macrophages we exposed LPS treated macrophages to RPT cell supernatant, which contained endogenous NPs. Our studies demonstrated that exogenous NPs had no effects on the NO production of cytokine/LPS stimulated PT cells.  However, the NPs increased the NO production of LPS treated macrophages via NPRs -A and -B, and the production of cyclic guanine monophosphate (cGMP) and subsequent activation of cGMP dependent protein kinase.  Paradoxically, endogenous NPs, contained within RPT cell supernatant, inhibited the NO production of LPS treated macrophages, suggesting that other RPT cell derived factors primed the macrophages to respond to the NPs by decreasing NO production. In conclusion our results suggest that NP mediated PT cell/macrophage interactions may regulate the inducible NO production of activated macrophages, but not PT cells, during tubulointerstitial inflammation.  PT cells may produce factors that facilitate NP mediated inhibition of macrophage.  NO synthesis as well as being sources of NPs.  Therefore NP mediated PT cell/macrophage interactions that inhibit macrophage NO synthesis may be an important protective mechanism during tubulointerstitial inflammation.

No description available.

The intrinsic characteristics of spontaneous, phasic activity in response to volume have revealed a period of inhibition following volume reduction termed the inhibitory phase. This is dependent upon the magnitude and duration of volume increase prior to decrease and is regulated by a distinct mechanism comprising of a combination of excitatory and inhibitory stimuli that may be co-ordinated by ganglia. It has also been found to be manipulated by antimuscarinics, nicotinic antagonists and purinergic agonists suggesting its underlying generation and regulation to be complex. The actions of hexamethonium and pancuronium upon phasic activity have suggested the presence of non-ganglionic nicotinic receptors involved in the modulation of spontaneous and volume induced activity; in addition to the role of ganglionic nicotinic receptors in co-ordinating the return of activity during the inhibitory phase. Purinoceptors were found to display a varied and complex response, both on pacemaker activity and upon the inhibitory phase. Both excitatory and inhibitory receptors were present; however, individual subtypes seemed to display a differing degree of functional relevance, varying between low and high volume. Though P2X and P2Y may have excitatory and inhibitory effects respectively, the overall effect of purinergic receptor stimulation upon phasic activity seems to be inhibitory. These experiments illustrate the mechanisms involved in the generation and co-ordination of spontaneous activity to be complex. It highlights novel mechanisms through which acetylcholine and adenosine triphosphate may be exerting an effect, and may account for the therapeutic actions of antimuscarinic medications. It also highlights potential mechanisms which may act as further therapeutic targets in the development of newer drugs for the treatment of OAB.

Diabetic and normal glomerular basement membranes have been examined chemically and immunologically for differences which could be pathognomonic of diabetes mellitus. Kidneys were obtained at post-mortem from diabetic subjects, normal subjects over sixty years and also from normal subjects under thirty years following fatal road accidents. Solubilisation of isolated glomeruli was effected by (a) enzymic digestion using collagenase and (b) chaotropic reagents; the solubilised components were identified by immunoelectrophoresis and polyacrylamide gel electrophoresis. A similarity of the antigenic components between all three groups was noted. Carbohydrate analysis of the glomerular basement membranes from diabetic and old normal groups aged above sixty years showed a decrease in sialic acid and glucose content with an associated increase in hexosamine, galactose and total hexose when compared with normal subjects aged below thirty years. The molar ratio of galactose/glucose was higher in the diabetic and old normal groups which, since the ratio of galactose to hydroxylysine was similar in all groups, indicates an increased number of hydroxylysine/galactose residues in diabetics and old normals. Amino acid analyses of glomeruli showed an increase in hydroxylysine, glycine and proline in diabetic and old normal groups only and there was also an associated decrease in lysine and histidine. The diabetic kidney was studied for the presence of antibody to basement membrane using immunofluorescence. The results showed an inherent fluorescence in diabetic and old normal kidneys and a deposition of immunoglobulin in some diabetic kidneys. Diabetic sera were also investigated using indirect immunofluorescence, but no antibody was detected. Further, leucocytes from diabetic patients were investigated in vitro for a cell-mediated response to basement membrane antigens using the leucocyte migration inhibition test. Inhibition of migration was not detected either with normal or diabetic glomerular basement membrane. The differences observed between diabetic and young normal glomeruli could be due to an acceleration of processes associated with age. In the light of the present findings investigation of the chemical and immunological differences between glomeruli from juvenile diabetics and young normal glomeruli should prove interesting.

In recent decades one of the major forces driving lower urinary tract research has been the aim of discovering the origins of bladder dysfunction and developing new therapeutic agents for treatment of conditions such as detrusor overactivity (DO). It is now becoming clear that the simplistic view of the control of detrusor function is far more complicated and involves complex interactions between various components of the bladder. With limited access to human tissue, animal models have been used for investigating the underlying mechanisms that control the detrusor function under pathological states. The aim of this project was to investigate the physiology and pathophysiology of bladder function in the streptozotocin (STZ)-induced diabetic rat model, 1, 4, 8 and 12-weeks post STZ injection. This was achieved by a combination of pharmacological, molecular biology and immunostaining techniques. Antimuscarinic drugs are the current pharmacological agents available for treatment of DO; however, there is much controversy surrounding the muscarinic mediated responses of the bladders from animal models of DO. Therefore, the muscarinic receptor mediated smooth muscle contractions were investigated in detrusor strips from the diabetic rat. An increased agonist mediated response was detected in detrusor strips from 1-week diabetic rats, which was mediated by M3-muscarinic receptor subtype. Basal spontaneous activity (SA) was detected in detrusor strips from control (nondiabetic) and diabetic but not in 12-week diabetic tissues. 1-week diabetic tissues showed increased amplitude of basal SA compared to all other groups. Since increased amplitude of SA of the bladder is thought to be associated with DO, the mechanisms that may be regulating these contractions were explored. SA could be induced/modulated by low concentrations of muscarinic receptor agonist carbachol (CCH). Once again, the amplitude of CCH-induced SA was significantly bigger in 1-week diabetic tissues compared to all other groups. No role for the mucosa in mediating the CCH-induced SA was demonstrated; however, the mucosa may have a role in 12-week diabetic tissues. The role of various potassium (K+) channels in modulating the SA was also investigated. Results demonstrated that opening of large conductance calcium activated potassium channels (BK Ca) reduced the amplitude of CCH-induced SA in both control (non-diabetic) and diabetic groups, whilst blocking these channels resulted in an increased basal SA in all groups except 12-week diabetic tissues. A decreased expression of BK Ca channel subunits' mRNA was detected in all diabetic bladders. Opening of ATP sensitive K+ channels (K ATP) only decreased the frequency of CCH-induced SA. Blocking K ATP channels had no effect on basal SA in all tissues. A reduced expression of K ATP channel subunits' mRNA was also detected in diabetic rat bladders. Recently a specialised type of cell, termed interstitial cells (ICs) has become the focus of research. It is postulated that they are involved in mediating the mechanosensory function of this organ. In this study, ICs were also identified in the rat urinary bladder using molecular biology and immunostaining. ICs were shown to play a role in mediating the CCH-induced SA of detrusors from rats, since their inhibition resulted in reduced SA in both control (non-diabetic) and diabetic tissues. Marked differences were seen between 1-week and 12-week diabetic rat bladders and it was concluded that 1-week diabetic rats can be used as a model of DO.In conclusion, the data presented in this thesis, indicates that complex mechanisms and physiological processes are present in the urinary bladder. It is clear that little is known about the detailed integrated physiology of the bladder wall and the structures involved in mediating the detrusor contractility.

No description available.

It has been postulated that the enzyme hyaluronidase facilitates water reabsorption in the renal collecting duct by reducing the intercellular mucopolysaccharide level and enhancing epithelial permeability. The present study was designed to examine the effects of enzyme inhibition upon certain renal and urinary compositional and structural changes which are normally associated with antidiuresis, inhibition was brought about by administration of antiserum to a purified extract of rat urinary hyaluronidase, raised in rabbits. In rats pretreated with antiserum antidiuretic stimuli failed to elevate the cortico-medullary solute concentration gradient. This was chiefly a reflection of elevated tissue water content by conparison with similarly treated animals which received normal serum. Following water deprivation for up to forty-eight hours, urinary volumes were significantly higher in groups receiving antiserum than in groups receiving normal serum. The medullary content of Liucopolysaccharide was estimated by measuring hexosa/aine release on hydrolysis with HOl, It was found that the level decreased during antidiuresis, but pretreatment with antir'Cruia abolished this effect. Pretreatment with antiserum abolished the caluria which noriDally accompanies hydropenia. Excretory levels of calcium between antiserum and normal serum pretreated groups differed significantly throughout the period of water deprivation. In the second twenty-four hour period excretory levels in both groups fell markedly. This was probably due to disturbances in normal body calcium balance resulting from the fall in dietary intake during water deprivation. Widening of intercellular spaces in collecting duct epithelium was observed in electron Jiicrographs of sections taken from kidneys of hydropenic rats. Pretreatnent with antiserum decreased both frequency and size of these spaces. In order to assess the specificity of the enzyme isolated from urine, antiserum to rat testicular hyaluronidase has been raised and its effects on the parameters refered to previously, examined. Results indicate that the testicular enzyme does not have significant effect on the renal concentrating process. These findings are consistant with the view that renal hyaluronidase plays a significant role in the process of urinary concentration.

Both acute and chronic steroidogenesis is regulated by adrenocorticotropin (ACTH), a principal regulator of the adrenal cortex. A number of studies have demonstrated that Steroidogenic Acute Regulatory (StAR) protein plays a crucial role in facilitating cholesterol transfer from the outer to the inner mitochondrial membrane where the first step of cholesterol conversion to steroid hormones occurs. This work has evaluated the relationships between the expression of StAR protein and signalling pathways of ACTH-induced steroidogenesis in primary cultures of bovine adrenal zona fasciculata (ZF) cells. A novel sheep anti-bovine peptide StAR polyclonal antibody has been characterised and optimised for Western immunoblotting. A newly formulated protocol based on enhanced chemiluminescence methodology provided a linear, reproducible and sensitive approach to detect and quantify StAR protein by molecular imaging analysis. The expression level of StAR protein after ACTH treatment of adrenal ZF cells showed that levels of StAR were insensitive to ACTH in freshly isolated cells due to the high initial levels of the protein. The cell responsiveness to ACTH was remarkable, however, after the basal levels of StAR protein diminished to relatively lower levels after 2 days of culture. Concentration-response curves demonstrated that, in general, increasing concentrations of ACTH resulted in increasing in cortisol output in parallel with increases of cyclic adenosine 3', 5'-monophsopathe (cAMP) production; the maximal effects of ACTH on cortisol and cAMP levels exhibited with 10^-8 M ACTH treatment after both 1 and 6 hr. Marked changes in StAR protein occurred at 6 hr. attaining a maximal level with 10^-8 M ACTH. Interestedly, at 1 and 6 hr the elevation of cortisol levels were significantly altered compared to the basal levels at 10^-12 M ACTH without any notable increase in cAMP. The time courses for ACTH treatment showed that at10^-8 M ACTH (a supraphysiological concentration) there was a strong correlation between cortisol and StAR protein induction, suggesting that newly synthesised StAR protein may be a major mediator for steroidogenesis.