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.

Adipose tissue is well-recognized as an endocrine organ which secretes a variety of bioactive molecules, including angiotensin II and its precursor angiotensinogen (Agt). There is mounting evidence linking the adipose renin-angiotensin system (RAS) and diet to obesity and obesity-related disorders. However, research addressing dietary regulation and function of adipose RAS is limited, and the specific mechanisms by which PUFAs modulate the endocrine function of adipose tissue remain largely unclear. There are several potential mechanisms that may mediate PUFA effects on Agt, including toll-like receptor signalling, prostaglandins or PPAR-gamma. Thus, we propose to investigate whether PUFAs differentially modulate Agt expression and secretion and to examine possible mechanisms by which PUFA alter Agt expression using the 3T3-L1 cell line. Differentiated 3T3-L1 adipocytes were treated with arachidonic acid (AA), eicosapentaenoic acid (EPA), AA + EPA, or vehicle (C) for 48 hours. Results showed a significant increase in intracellular Agt protein following treatment with PUFAs. Agt secretion, however, was only increased by AA. Interestingly, there is a dose-dependent decrease in Agt protein levels by EPA suggesting that a minimum concentration of n-3 PUFAs is required to elicit an Agt response. Agt mRNA levels were measured by RT-PCR and results showed a significant increase in Agt mRNA in response to treatment with AA but not EPA. These findings suggest that Agt regulation by PUFAs is complex and occurs both post-transcriptionally and post-translationally. Changes in mRNA stability may account for the observed effects of PUFAs. Adipocytes were treated with the transcriptional inhibitor actinomycin D (Act D) and Agt mRNA expression was measured over time. Total RNA was also measured at each time point to ensure that Act D treatment was effectively decreasing transcription. Agt mRNA expression was not significantly altered by treatment with EPA while treatment with AA increased Agt mRNA levels. These results suggest that Agt mRNA stability is differentially increased by n-6 but not n-3 PUFAs. Although there are clear effects of AA on Agt secretion and mRNA stability, the signaling pathways mediating this response remain to be determined, and additional studies are necessary to further dissect the underlying mechanisms of this regulation.

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.

No description available.

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.

Despite years of research, the role that hypovolemia plays in orthostatic intolerance after head down bed rest (BR) and spaceflight remains unclear. Additionally, the efficacy of oral saline countermeasures, employed in an attempt to restore plasma volume (PV) after BR is questionable. Several previous studies have suggested that a new homeostatic set point is achieved in space or during BR, making attempts to restore PV temporary at best. We tested the hypotheses that one day of BR would induce a transient increase in PV followed by hypovolemia and new hormonal balance; that a salt tablet and water fluid loading (FL) countermeasure would be ineffective in restoring PV; and also that the FL would not attenuate the exaggerated hormonal responses to orthostatic stress that are expected after 28hr of BR. Plasma volume, serum sodium and osmolarity, and plasma ANP, AVP, renin, angiotensin II, aldosterone, and catecholamines were measured in nine male subjects undergoing 5 different protocols (28hr Bed Rest without Fluid Loading = 28NFL, 28hr Bed Rest with Fluid Loading = 28FL, 4hr Seated Control = 4NFLS, 4hr Seated Control with Fluid Loading = 4FLS, and 4hr Bed Rest = 4BR) in a randomized repeated measures design. The FL countermeasure was 15 ml/kg of body weight of water with 1g of NaCl per 125ml of water. Orthostatic testing by lower body negative pressure (LBNP) was performed before and after all protocols. In agreement with our first hypothesis, we observed transient reductions in renin, angiotensin II, and aldosterone, which after 25.5hr were restored to baseline, slightly augmented, and suppressed, respectively. Also after 25.5hr, PV was reduced in the 28hr BR protocols and was not restored in 28FL; however, the FL protocol increased PV during 4FLS. We additionally observed augmented renin and aldosterone responses, as well as generally elevated angiotensin II after 28NFL, but not after 28FL or any of the 4hr protocols. Furthermore, no changes in plasma norepinephrine responses to LBNP were documented from Pre-Post test in any protocol. Our results indicate that: 1) PV is reduced after short term BR and is not restored by an oral FL; 2) renin-angiotensin-aldosterone system (RAAS) responses to orthostatic stress are augmented after 28hr of BR and the amplified response can be abrogated by FL; and 3) plasma norepinephrine responses during orthostatic stress are not affected by BR or FL, suggesting that RAAS activity may be modulated by FL independently of sympathetic activity and PV during orthostasis after bed rest.

Despite years of research, the role that hypovolemia plays in orthostatic intolerance after head down bed rest (BR) and spaceflight remains unclear. Additionally, the efficacy of oral saline countermeasures, employed in an attempt to restore plasma volume (PV) after BR is questionable. Several previous studies have suggested that a new homeostatic set point is achieved in space or during BR, making attempts to restore PV temporary at best. We tested the hypotheses that one day of BR would induce a transient increase in PV followed by hypovolemia and new hormonal balance; that a salt tablet and water fluid loading (FL) countermeasure would be ineffective in restoring PV; and also that the FL would not attenuate the exaggerated hormonal responses to orthostatic stress that are expected after 28hr of BR. Plasma volume, serum sodium and osmolarity, and plasma ANP, AVP, renin, angiotensin II, aldosterone, and catecholamines were measured in nine male subjects undergoing 5 different protocols (28hr Bed Rest without Fluid Loading = 28NFL, 28hr Bed Rest with Fluid Loading = 28FL, 4hr Seated Control = 4NFLS, 4hr Seated Control with Fluid Loading = 4FLS, and 4hr Bed Rest = 4BR) in a randomized repeated measures design. The FL countermeasure was 15 ml/kg of body weight of water with 1g of NaCl per 125ml of water. Orthostatic testing by lower body negative pressure (LBNP) was performed before and after all protocols. In agreement with our first hypothesis, we observed transient reductions in renin, angiotensin II, and aldosterone, which after 25.5hr were restored to baseline, slightly augmented, and suppressed, respectively. Also after 25.5hr, PV was reduced in the 28hr BR protocols and was not restored in 28FL; however, the FL protocol increased PV during 4FLS. We additionally observed augmented renin and aldosterone responses, as well as generally elevated angiotensin II after 28NFL, but not after 28FL or any of the 4hr protocols. Furthermore, no changes in plasma norepinephrine responses to LBNP were documented from Pre-Post test in any protocol. Our results indicate that: 1) PV is reduced after short term BR and is not restored by an oral FL; 2) renin-angiotensin-aldosterone system (RAAS) responses to orthostatic stress are augmented after 28hr of BR and the amplified response can be abrogated by FL; and 3) plasma norepinephrine responses during orthostatic stress are not affected by BR or FL, suggesting that RAAS activity may be modulated by FL independently of sympathetic activity and PV during orthostasis after bed rest.

Adipose tissue is well-recognized as an endocrine organ which secretes a variety of bioactive molecules, including angiotensin II and its precursor angiotensinogen (Agt). There is mounting evidence linking the adipose renin-angiotensin system (RAS) and diet to obesity and obesity-related disorders. However, research addressing dietary regulation and function of adipose RAS is limited, and the specific mechanisms by which PUFAs modulate the endocrine function of adipose tissue remain largely unclear. There are several potential mechanisms that may mediate PUFA effects on Agt, including toll-like receptor signalling, prostaglandins or PPAR-gamma. Thus, we propose to investigate whether PUFAs differentially modulate Agt expression and secretion and to examine possible mechanisms by which PUFA alter Agt expression using the 3T3-L1 cell line.Differentiated 3T3-L1 adipocytes were treated with arachidonic acid (AA), eicosapentaenoic acid (EPA), AA + EPA, or vehicle (C) for 48 hours. Results showed a significant increase in intracellular Agt protein following treatment with PUFAs. Agt secretion, however, was only increased by AA. Interestingly, there is a dose-dependent decrease in Agt protein levels by EPA suggesting that a minimum concentration of n-3 PUFAs is required to elicit an Agt response. Agt mRNA levels were measured by RT-PCR and results showed a significant increase in Agt mRNA in response to treatment with AA but not EPA. These findings suggest that Agt regulation by PUFAs is complex and occurs both post-transcriptionally and post-translationally.Changes in mRNA stability may account for the observed effects of PUFAs. Adipocytes were treated with the transcriptional inhibitor actinomycin D (Act D) and Agt mRNA expression was measured over time. Total RNA was also measured at each time point to ensure that Act D treatment was effectively decreasing transcription. Agt mRNA expression was not significantly altered by treatment with EPA while treatment with AA increased Agt mRNA levels. These results suggest that Agt mRNA stability is differentially increased by n-6 but not n-3 PUFAs. Although there are clear effects of AA on Agt secretion and mRNA stability, the signaling pathways mediating this response remain to be determined, and additional studies are necessary to further dissect the underlying mechanisms of this regulation.

No description available.

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.