Angiotensinogen is a blood protein that plays a critical role in the regulation of blood pressure in the body. This protein exists in two forms, oxidised and reduced, determined by the presence or absence of a disulfide bridge between Cys 18 and Cys 138. The ratio of oxidised to reduced angiotensinogen is 60:40 in the blood of healthy individuals - an equilibrium that is disrupted in women who develop pre-eclampsia, leading to a higher proportion of oxidised angiotensinogen in the blood. Pre-eclampsia, one of the leading causes of premature births, is a severe and potentially fatal pregnancy condition characterised by the sudden onset of symptoms such as high blood pressure and proteinuria typically during the third trimester. This condition is responsible for an estimated 550,000 deaths globally each year, and with no available treatment or cure other than early delivery of the child, there is a desperate need for a reliable and predictive diagnostic test for this condition. Can we use angiotensinogen as a biomarker for the early diagnosis of pre-eclampsia? Being able to distinguish between reduced and oxidised angiotensinogen and determine the relative amounts of each in blood samples would be of a huge diagnostic value for this condition. This thesis outlines the expression and purification of recombinant human angiotensinogen in Escherichia coli, and the development of an antibody-based SPR assay for angiotensinogen that was subsequently used to probe whether reduced and oxidised angiotensinogen can be distinguished experimentally. The assay developed was sensitive and reproducible, and demonstrated that the reduced and oxidised forms can be distinguished experimentally. The antibody bound the two forms with differential affinity, due to differences in both the association and dissociation rates of the two forms with the monoclonal antibody. Finally, in an attempt to further elucidate the differences between the two redox states of angiotensinogen, molecular dynamic simulations were carried out on angiotensinogen in the presence or absence of the disulfide bond between Cys 18 and Cys 138. These simulations revealed some quite striking differences in the dynamics between the two forms. Reduced angiotensinogen was found to be more dynamic in regions critical for binding to renin, providing a possible explanation for the reported differential affinity that renin displays for the two forms.1 Thus, reduced and oxidised angiotensinogen show some quite distinct differences and can be distinguished in an SPR-based assay, highlighting their potential for use as a biomarker in a diagnostic bioassay.

In spite of significant progress in our knowledge of mechanisms that control blood pressure, our understanding of the pathogenesis of hypertension, its genetics, and population efforts to control blood pressure, hypertension remains the leading risk factor for mortality worldwide. It’s estimated that 1 out of every 3 adults has hypertension. Hypertension is a major risk factor for cardiovascular disease and stroke, and is considered a primary or contributing cause of death to more than 2.4 million US deaths each year. Although spontaneous hypertension has been the subject of substantial research, many critical questions remain unanswered. To investigate mechanisms underlying spontaneous hypertension, a unique rodent breeding approach was used to isolate nuclear and mitochondrial genes contributing to the disease. By diluting the nuclear genome of the Spontaneously Hypertensive Rat on a normotensive Brown Norway background while maintaining the SHR mitochondrial genome, I investigated both intrinsic and extrinsic mechanisms of the kidney and its relationship to hypertension. Chapter 2 documents the dominance of the hypertensive phenotype in our rodent colony, despite the dilution of the nuclear genome of the SHR. Chapter 3 presents data indicating that the renin-angiotensin system, particularly the location and abundance of the AT1 receptor may play an important role in the manifestation of spontaneous hypertension. Chapter 4 presents that rats in our rodent colony exhibited normal pressure-natriuresis and kidney function; however, hypertensive rats had a reduced ability to sense orally ingested sodium chloride, thus necessitating chronic elevations of arterial pressure in order to maintain sodium balance. This chronic pressure-natriuresis relationship shifts the renal function curve to the right, thus sustaining elevated blood pressure. Chapter 5 presents data that genes important for oxidative phosphorylation may play a critical role in the development of hypertension. Both nuclear and mitochondrial oxidative phosphorylation genes were downregulated in hypertensive rats compared with normotensive rats. Data presented in every chapter highlights the importance of the kidney in the pathogenesis of hypertension. Humoral, genetic and genomic mechanisms of the kidney appear to play a dominant role in the development and maintenance of the disease.

Objectives: The aim of this study was to determine associations between physical activity (PA), blood pressure (BP) and renin in urban black Africans. Methods: The study sample included 137 urban African males (N=68) and females (N=69) (aged 41.53 ± 8.13 and 44.16 ± 7.37 years, respectively), from the North West Province, South Africa. Anthropometric measurements, ambulatory blood pressure and energy expenditure were determined. Actical® accelerometers were used to determine energy expenditure (METS) over a 24 hour period. Fasting blood samples were used to determine fasting blood glucose, serum cotinine (COT), gamma–glutamyl transferase (GGT) and plasma renin. Results: A greater percentage (64%) of African males were hypertensive compared to African females (33.33%). SBP (p<0.001) and DBP (p<0.001) were significantly higher in males than females. Female subjects were more obese (32.00±7.75 kg/m2) whereas males demonstrated an overweight status (27.28±5.86kg/m2). Male subjects displayed overall higher lifestyle risks (BP, smoking, alcohol consumption, HIV–status) than females. Multivariate regression analyses demonstrated an inverse relationship between BP and renin in both males and females, but no associations existed between renin and physical inactivity. Conclusion: PA appeared not to buffer elevated blood pressure in this specific African sample, as no significant associations supported this hypothesis. The results confirm that black Africans display lower renin levels associated with elevated blood pressure. Furthermore, low renin and physical inactivity was not related to indicate elevated BP through elevated SNS activity. / Thesis (M.Sc. (Biokinetics))--North-West University, Potchefstroom Campus, 2012.

The Angiotensin II Type 1 (AT1) receptor is the main receptor responsible for the effects of the renin-angiotensin system, and its expression pattern is altered in several diseases. [11C]Methyl-Losartan has been developed based on the clinically used AT1 receptor antagonist Losartan. The aim of this work is to characterize the pharmacokinetics, repeatability and reliability of measurements, binding specificity and selectivity of [11C]Methyl-Losartan in rats using in vivo small animal positron emission tomography (PET) imaging, ex vivo biodistribution and in vitro autoradiography methods. Also, we aim to measure the presence of metabolites in the kidney and plasma using high-performance liquid chromatography. We have demonstrated in vivo that [11C]Methyl-Losartan is taken up in the AT1 receptor-rich kidneys and that it is displaceable by selective AT1 receptor antagonists. Using ex vivo biodistribution, we have confirmed these results and demonstrated that [11C]Methyl-Losartan binds selectively to the AT1 receptor over the AT2, Mas and β-adrenergic receptors. In vitro autoradiography results confirmed these renal binding selectivity studies. [11C]Methyl-Losartan was also shown to have one and two C-11 labeled metabolites in the plasma and kidneys, respectively. In conclusion, [11C]Methyl-Losartan is a promising agent for studying the AT1 receptor in rat models with normal and altered AT1 receptor expression using small animal PET imaging.

Pea protein hydrolysate (PPH) has antihypertensive effects and prostanoids have been implicated in renal diseases. To investigate the role of PPH and prostanoids on renal and cardiovascular effects in cardio-renal disease, normal and diseased Han:SPRD-cy rats were given diets containing either 0, 0.5% or 1% PPH for 8 weeks. At termination, diseased rat kidneys displayed increased renal cyst growth, fibrosis, plasma creatinine and lower monocyte chemoattractant protein-1. Diseased rats also exhibited left ventricular (LV) hypertrophy, elevated systolic and diastolic blood pressures and LV end diastolic and systolic pressures. Four of five prostanoids were elevated in diseased rat kidneys. PPH attenuated systolic blood pressure, but not other components of the cardio-renal syndrome. PPH also increased select prostanoids in normal and diseased rats. Thus, dietary PPH attenuates hypertension in the Han:SPRD-cy rat, but does not ameliorate other components of disease, possibly due to increased prostanoid effects or an insufficient treatment length.

Objectives: The aim of this study was to determine associations between physical activity (PA), blood pressure (BP) and renin in urban black Africans. Methods: The study sample included 137 urban African males (N=68) and females (N=69) (aged 41.53 ± 8.13 and 44.16 ± 7.37 years, respectively), from the North West Province, South Africa. Anthropometric measurements, ambulatory blood pressure and energy expenditure were determined. Actical® accelerometers were used to determine energy expenditure (METS) over a 24 hour period. Fasting blood samples were used to determine fasting blood glucose, serum cotinine (COT), gamma–glutamyl transferase (GGT) and plasma renin. Results: A greater percentage (64%) of African males were hypertensive compared to African females (33.33%). SBP (p<0.001) and DBP (p<0.001) were significantly higher in males than females. Female subjects were more obese (32.00±7.75 kg/m2) whereas males demonstrated an overweight status (27.28±5.86kg/m2). Male subjects displayed overall higher lifestyle risks (BP, smoking, alcohol consumption, HIV–status) than females. Multivariate regression analyses demonstrated an inverse relationship between BP and renin in both males and females, but no associations existed between renin and physical inactivity. Conclusion: PA appeared not to buffer elevated blood pressure in this specific African sample, as no significant associations supported this hypothesis. The results confirm that black Africans display lower renin levels associated with elevated blood pressure. Furthermore, low renin and physical inactivity was not related to indicate elevated BP through elevated SNS activity. / Thesis (M.Sc. (Biokinetics))--North-West University, Potchefstroom Campus, 2012.

The renin-angiotensin-aldosterone system (RAAS) plays an essential role in maintaining plasma volume and arterial blood pressure by regulating angiotensin II levels. Dysregulation of the RAAS can result from an underlying disorder that results in a severe and untreatable form of hypertension, known as secondary hypertension. Measurement of plasma renin activity is a commonly employed method of diagnosing secondary hypertension. Plasma renin activity is quantified by determining the amount of angiotensin I generated through the enzymatic cleavage of angiotensinogen by renin. Radioimmunoassay is routinely used to measure plasma renin activity, however there are limitations to the method. With the prevalence of hypertension on the rise, there is need for a more accurate and rapid method of assessing the RAAS for diagnostic purposes and therapeutic monitoring. Multiplexed measurement of angiotensin I and angiotensin II would provide comprehensive understanding of the RAAS by determining dysregulation in the production of either molecule. In this thesis, the relationship between endogenous angiotensin I concentrations and plasma renin activity are studied in order to examine the research hypothesis that measurement of angiotensin I concentration correlates with plasma renin activity and whether this may provide a more accurate and rapid method of screening for hypertension when multiplexed with angiotensin II. To overcome the current limitations of radioimmunoassay for measuring plasma renin activity, a mass spectrometric-based method was developed to measure angiotensin I and plasma renin activity. Evaluation of the assay against radioimmunoassay demonstrates that the assay is reproducible and provides a linear response over a diagnostically relevant concentration range. Comparison of endogenous levels of angiotensin I with normal plasma renin activity levels show a correlation in this study (R=0.74). Comparison of plasma renin activity values by radioimmunoassay and iMALDI also show correlation (R=0.98), indicating that the iMALDI assay may provide an improved method for diagnosing secondary hypertension.

In 2008 the global prevalence of hypertension [high blood pressure (BP), systolic ≥140 mmHg and/or diastolic ≥90 mmHg] was around 40% in adults > 25 yrs of age, according to the 2013 WHO statistics. Hypertension is a major risk factor for myocardial infarction, heart failure and stroke. Currently, around 20% of the Canadian population is affected by hypertension. Hypertension is more closely associated with diabetes. More than two thirds of people with diabetes have hypertension, alongwith increased activity of the renin angiotensin aldosterone (RAAS) system. The RAAS plays a major role in maintaining fluid balance, vascular tone and BP. The components of the RAAS include the hormone renin, which cleaves angiotensinogen, a circulating inactive peptide into angiotensin I. Angiotensin converting enzyme (ACE) converts angiotensin I into the active peptide angiotensin II (Ang II). Ang II causes vasoconstriction, sodium reabsorption from the kidney tubules and also release of the hormone, aldosterone, from the adrenal cortex. The epidemic of hypertension, diabetes and obesity is widely attributed to a high carbohydrate diet, containing mainly high fructose corn syrup and sucrose. However, the underlying molecular mechanisms are far from clear. A high fructose diet increases BP in Sprague-Dawley (SD) rats; along with elevated plasma and aortic levels of methylglyoxal (MG). MG is a reactive dicarbonyl compound mainly formed as an intermediate during glycolysis. Small amounts of MG are also formed during amino acid (threonine) and fatty acid metabolism. MG reacts with certain proteins to form irreversible advanced glycation end products (AGEs). MG has high affinity for arginine, lysine and cysteine. Plasma MG levels are increased in hypertensive rats and diabetic patients. However, it is not yet clear whether MG is the cause or effect of hypertension. Moreover, safe and specific MG scavengers are not available. The aim of the project was to determine the effect of MG and a high fructose diet on the RAAS and the BP in male SD rats. The hypothesis that L-arginine, and its inactive isomer D-arginine, can efficiently scavenge MG in vitro, was also tested. Male SD rats were treated with a continuous infusion of MG with a subcutaneous minipump for 4 weeks, or with a high fructose diet (60% of total calories) for 16 weeks. We also used isolated aortic rings from 12 week old normal male SD rats to study endothelial function. Organs / tissues, cultured human umbilical vein endothelial cells (HUVECs) and vascular smooth muscle cells (VSMCs) were used for molecular studies. HPLC, Western blotting and Q-PCR were used to measure MG, reduced glutathione (GSH), proteins and mRNA, respectively. siRNA for angiotensinogen and the receptor for advanced glycation endproducts (RAGE) were used to study mechanisms. MG treated rats developed a significant increase in BP and plasma levels of aldosterone, renin, angiotensin and catecholamines. MG level, and protein and mRNA for angiotensin, AT1 receptor, adrenergic α1D receptor and renin were significantly increased in the aorta and/or kidney of MG treated rats, a novel finding. Alagebrium, a MG scavenger and AGEs breaker, attenuated the above effects of MG. Treatment of cultured VSMCs with MG or high glucose (25mM) significantly increased cellular MG, and protein and mRNA for nuclear factor kappa B (NF-κB), angiotensin, AT1 and α1D receptors, which were prevented by inhibition of NF-κB, and by alagebrium. Silencing of mRNA for RAGE prevented the increase in NF-kB induced by MG. Silencing of mRNA for angiotensinogen prevented the increase in NF-κB, angiotensin, AT1 and α1D receptors’ protein. Fructose treated rats developed a significant increase in BP. MG level and protein and mRNA for angiotensin II, AT1 receptor, adrenergic α1D receptor and renin were significantly increased, whereas GSH levels were decreased, in the aorta and/or kidney of fructose fed rats. The protein expression of the receptor for AGEs (RAGE) and NF-κB were also significantly increased in the aorta of fructose fed rats. MG treated VSMCs showed increased protein for angiotensin II, AT1 receptor, and α1D receptor. The effects of fructose and MG were attenuated by metformin, a MG scavenger and AGEs inhibitor. In experiments to test the MG scavenging action of arginine, both D-arginine and L-arginine prevented the attenuation of acetylcholine-induced endothelium-dependent vasorelaxation by MG and high glucose. However, the inhibitory effect of the NOS inhibitor, Nω-nitro-L-arginine methyl ester, on vasorelaxation was prevented only by L-arginine, but not by D-arginine. MG and high glucose increased protein expression of arginase, a novel finding, and also of NADPH oxidase 4 and NF-κB, and production of reactive oxygen species in HUVECs and VSMCs, which were attenuated by D- and L-arginine. However, D- and L-arginine did not attenuate MG and high glucose-induced increased arginase activity in VSMCs and the aorta. D- and L-arginine also attenuated the increased formation of the MG-specific AGE, Nε-carboxyethyl lysine, caused by MG and high glucose in VSMCs. In conclusion, MG activates NF-κB through RAGE and thereby increases renin angiotensin levels, a novel finding, and a probable mechanism of increase in BP. There is a strong association between elevated levels of MG, RAGE, NF-κB, mediators of the RAAS and BP in high fructose diet fed rats. Arginine attenuates the increased arginase expression, oxidative stress, endothelial dysfunction and AGEs formation induced by MG and high glucose, by an endothelial NOS independent mechanism.

This thesis reports the effects of an altered intrauterine environment on the offspring???s renal, cardiovascular and renin angiotensin systems. After a midgestational asphyxial episode in fetal sheep (30 min total umbilical cord occlusion at 90 days; term 150 days) the hydrops that resulted had not completely resolved by 130 days. While the heart and kidneys were apparently unaffected, the brain and lung weights were 37% and 50% lower than sham values, respectively and there were joint contractures. The effects of maternal renal disease on the offspring were investigated. Although in utero fetuses of subtotally nephrectomised ewes (STNx) had altered urine flow rates, sodium excretion, haematocrits, plasma chloride and plasma renin levels, by 1-2 weeks after birth these values in the lambs (STNxL) were similar to controls (ConL) under baseline conditions. Body weight and the weights of most organs were similar, including the kidney, in which glomerular number was normal. In the neonatal period, the lambs were subjected to four challenges: furosemide (2 mg/kg intravenous bolus), infusion of angiotensin II and phenylephrine, intravenous infusion of 0.15M saline (50 ml/kg over 30 min) and haemorrhage (20% estimated blood volume over 10 min). These challenges revealed evidence of programming of several aspects of the renal, cardiovascular and renin angiotensin systems in the STNx offspring. As young adults at 6 months of age, male and female offspring of STNx ewes were normotensive and had normal renal function. On a high salt diet (HSD, 0.17M NaCl in 8L water for 5-7days), female offspring of both groups did not become hypertensive. However, the STNx offspring must have retained salt and water as plasma sodium was increased and haematocrit was decreased. In the STNx offspring only, there was a relationship between glomerular filtration rate (GFR) and mean arterial pressure, indicating an inability to maintain a constant GFR in response to changes in arterial pressure.

Bibliography : leaves 168-189. / xix, 197 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Examines the role of renin-angiotensin and sympathetic nervous systems in hypertension development in the spontaneously hypertensive rat. / Thesis (Ph.D.)--University of Adelaide, Dept. of Clinical and Experimental Pharmacology, 1995?