A collection of salt-sensitive vacuolar (ssv) Saccharomyces cerevisiae mutants were selected for analysis in an attempt to reveal components of the osmosensing signal transduction pathway. A previous screen of these mutants had been used to select those with an impaired glycerol response to salt stress. In this study the glycerol-3-phosphate dehydrogenase activity was measured in all the strains which showed reduced glycerol accumulation to demonstrate a corresponding low enzyme activity which could be caused by lack of signalling through the high osmolarity glycerol (HOG) response pathway. However, enzyme activity was found to be impaired in only one of the strains tested. This demonstrated that the measurement of glycerol accumulation is not a particularly useful screening method for defects in the HOG pathway. The activity of the promoter of CTTl, another stress-responsive gene, was measured in selected ssv strains using a lacZ reporter gene attached to the CTTl promoter stress response element. This gave further information about the stress-responsiveness in the strains tested. CTTl promoter activity did not correlate with GPDH activity in all of the strains tested. As CTTl is subject to control by more than one type of stress the results imply that in at least one of the strains another stress response may be impaired. The VACl homologue (VACIH) on chromosome XIV was characterised as a candidate for one of the 55V genes, 55W7. Although it was demonstrated not to be 55W 7, a role for VACIH in vacuolar protein sorting was discovered. The Δvaclh strain also displayed class E vacuolar morphology. Sequence analysis and complementation experiments demonstrated that VACIH is identical to VPS27.

The World Health Organization (WHO) states that iron deficiency (ID) is the preeminent global micronutrient deficiency. Maternal ID is linked to cardiovascular disease and hypertension in offspring, and yet the impact of ID on the kidney is not known. We characterized the impact of ID on circulating and tissue levels of iron and renal function. Seven and ten week old male and female Wistar rats were fed either control (270ppm) or low iron (3ppm) diet for ten weeks. Total, non-heme and heme iron levels in liver, kidney cortex and kidney medulla were measured following the dietary period. Hematocrit decreased most in males regardless of age (Male 7-17wk: 47%→ 31%; 10-20wk: 47%→ 33.5%; Female 7-17wk: 47%→ 34%; 10-20wk: 47%→ 39%). Dietary ID markedly decreased liver and kidney cortex non-heme iron in both males and females (Female: liver-178±25 to 21±7 ppm; kidney cortex-51±9 ppm to 10±1 ppm; Male: liver-102±18 ppm to 11±1 ppm; kidney cortex 36±14 to 15±8 ppm). In contrast, non-heme iron in the kidney medulla was not significantly decreased Secondly, in order to determine the impact of ID on renal function, blood pressure was monitored using radio-telemetry starting at six weeks of age (~175g body weight). Dietary salt challenge (5 days Low5 days HighNormal) was administered to all animals (n=16) starting at eight weeks of age. At ten weeks rats were assigned to either control (225pm) or low (3ppm) iron diet. Dietary salt challenge was repeated at 13 and 18 weeks of age respectively. Despite significant lowering of hematocrit (Control 45%  Low iron 38.6%) hemodynamic changes were minimal, in that, although blood pressure was lowered following ten weeks of dietary iron restriction, blood pressure did not change in response to dietary salt (Control MAP:105.0 ± 2.5mmHg; ID MAP:100.6 ± 3.2mmHg ). Collectively the tissue and functional analyses demonstrate that the body adapts to lowering of tissue iron supply with ID. The relative sparing of non-heme iron in the kidney medulla suggests that iron in this region of the kidney may be spared because of its importance in the systems responsible for regulating fluid and sodium balance. / Thesis (Master, Pharmacology & Toxicology) -- Queen's University, 2007-07-18 12:00:09.853

Under normotensive conditions, the progressive loss of renal function in diabetes mellitus is very slow. Since hypertension accelerates many forms of renal disease, we assessed the progression of nephropathy in Streptozotocin-induced type 1 diabetes mellitus under renin-mediated hypertension condition. We investigated the diabetic “salt paradox” as a modifiable susceptibility factor for renal damage. Since hyperfiltration occurs in early diabetes, the reduction of glomerular filtration rate due to an increased salt intake could be mediated by increased tubuloglomerular feedback sensitivity. We compared intact-hypertensive versus diabetic-hypertensive Long-Evans rats under normal and increased salt intake, 1 and 2.5% by weight of food eaten, respectively. Weekly 24-h blood pressure records were acquired by telemetry during the six months of the experiment. Target mean blood glucose of ~ 25 mmol/L was maintained by suboptimal insulin implants. Systolic blood pressure increased after induction of hypertension but was not affected by diabetes or increased salt intake, either alone or together. Autoregulation was highly efficient in both intact and diabetic rats. Nephropathy was scored by histology in the clipped and non-clipped kidneys at the end of the protocol. The non-clipped kidney, which was exposed to hypertension, showed a linear pressure-dependent glomerular injury in both intact and diabetic rats. The best fit line describing the linear relationship between pressure load and injury was shifted toward lower blood pressure in diabetic rats. Over the time course of our experiments, injury was entirely pressure dependent in intact and diabetic rats. Diabetes mellitus increased the susceptibility of the kidney to injury, but independent of blood pressure. Increased salt intake affected neither blood pressure nor renal susceptibility to hypertensive injury. / Graduate

No description available.

Background: Hypertension, a leading risk factor for cardiovascular disease, exhibited in 17.7% of the Canadian population, and attributed to 13% of world mortality, is influenced by both the environment and genetics. Salt sensitivity is described at higher rates in the hypertensive population. The NEDD4-like (NEDD4L) protein is important in sodium reabsorption and has been implicated in essential hypertension and salt sensitivity. Objectives: Two variations (rs4149601/rs2288774) found in NEDD4L have been associated with salt sensitivity and hypertension; a third (rs576416) is in linkage disequilibrium with rs4149601. The purpose of this study is to assess the relationship between the NEDD4L rs4149601, rs2288774, and rs576416 single nucleotide polymorphisms with sodium and age on blood pressure (BP). Methods: Canadian hypertensive patients were recruited through the University of Ottawa Heart Institute, with genotyped data from Leuven, Belgium, and the DNA of subjects from Warsaw, Poland also included in the study. Eligible subjects were studied off anti-hypertensive medications. Daytime BP was measured using 24hr ambulatory BP monitoring in 662 Caucasian hypertensives (BP ≥130/85 mmHg). 24hr urine Na+ was collected. DNA from Canada and Poland was genotyped on the GeneTitan Affymetrix Axiom platform and through TaqMan MGB probe-based RT-PCR, while the Belgium samples were analyzed on Illumina 1M-duo arrays. Simple and multivariate linear regression modelling with SAS 9.4.0 was used for genotypic comparisons affecting BP, combined with age and corrected urine sodium. Results: The three hypertensive populations were significantly different (P<0.05) across all demographic and clinical measures, even when stratified by sex. The Polish and female hypertensives from Canada and Belgium were removed from the analysis for lacking the general populations’ trend of increasing BP with age. Multiple linear regressive modelling found a significant association (Pmodel=0.0034) of rs4149601 GA (P=0.0129) and GG (P=0.0082), with age and urine sodium, on SBP in the Belgium male hypertensives (n=273). No significant models analyzing the association of rs576416 and rs2288774 with BP in the Belgium population were found. In the Canadian hypertensive population (n=120) no association on the discrete analyses of the rs4149601, rs576416, and rs2288774 genotypes were found; however the combination of the GG rs4149601 and AA rs576416 (β=0.021, P=0.03) and the GG rs4149601 and CC rs2288774 (β=0.020, P=0.04) genotypes showed significant associations with BP in borderline significant models (P=0.055 and P=0.094 respectively), when analyzed with urine sodium levels and age. Conclusions: A significant influence of the rs4149601 G-allele, with urine sodium and age, was found to be associated with an increase in BP in the Belgium males. Multiple linear modelling describing borderline significant findings in the interaction of rs4149601 with rs576416, and rs4149601 with rs2288774 in Canadian male hypertensives suggests of the possible synergism between polymorphisms and development of salt sensitive hypertension. Future research could evaluate the role of NEDD4L on the sex differences in early-onset salt-sensitive hypertension.

Prostaglandin E2 (PGE2), a metabolite of arachidonic acid, plays a role in water and sodium reabsorption in the collecting duct of the kidney. The collecting duct is responsible for the fine tuning of water and electrolytes. Only a small fraction of the filtered water and sodium is reabsorbed in the collecting duct, a fraction crucial to the regulation of water and electrolyte balance. This current study addresses the role of EP1, one of four PGE2 receptors, in the collecting duct. It is well documented that PGE2 inhibits sodium and water reabsorption in the collecting duct, however the exact mechanism is still debated. To determine whether the EP1 receptor mitigates AngII renal effects, an in vivo study was performed with EP1-/- mice. Global EP1-/- knockout mice were crossed with a renin overexpressing mouse line (herein denoted as “Ren”) and subjected to a high salt (HS) and low salt (LS) diet. Ren mice displayed an 11mmHg increase in systolic blood pressure (BP) on a HS diet and a decrease in BP of 14mmHg on a LS diet compared to the normal salt (NS) diet. Ren EP1-/- mice did not display a significant increase or decrease in BP on a HS or LS diet. On a LS diet, Ren EP1-/- displayed a drop in urine osmolarity (1641 mOsm/ kgH2O) vs. wild type (WT) mice (2107 mOsm/ kgH2O), consistent with increased sodium reabsorption. Narrowing in on the collecting duct, Ren EP1-/- mice had enhanced αENaC levels compared to Ren mice. In ex vivo microperfusion experiments, EP1-/- tubules show no response to PGE2 in the presence of AVP, whereas PGE2 inhibits AVP induced water reabsorption in WT mice. An increase in αENaC membrane accumulation due to EP1 gene ablation results in increased sodium reabsorption subsequently leading to a rise in BP. This contributes to the lack of salt sensitivity in EP1-/- mice. Overall, the EP1 receptor in the collecting duct represents a potential therapeutic target for the treatment of hypertension.

<p>Hydronephrosis, due to ureteropelvic junction obstruction, is a common condition in infants with an incidence of approximately 0.5-1%. During the last decade, the surgical management of non-symptomatic hydronephrosis has become more conservative, and the long-term physiological consequences of this new policy are unclear. The overall aim of this thesis was to determine whether there is a link between hydronephrosis and the development of hypertension. Hydronephrosis was induced by partial ureteral obstruction in 3-week old rats or mice. In the adult animals, blood pressure was measured telemetrically during different sodium conditions and the renal function was evaluated. Both species developed salt-sensitive hypertension and histopathological changes (i.e. fibrosis, inflammation, glomerular and tubular changes) that correlated with the degree of hydronephrosis. An abnormal renal excretion pattern with increased diuresis and impaired urine concentrating ability was observed in hydronephrosis. The mechanisms were primarily located to the diseased kidney, as relief of the obstruction attenuated blood pressure and salt-sensitivity. Increased renin angiotensin system activity, due to ureteral obstruction, might be involved in the development but not necessary the maintenance of hypertension. Hydronephrotic animals displayed reduced nitric oxide availability, which might be due to increased oxidative stress in the diseased kidney. Renal nitric oxide deficiency and subsequent resetting of the tubuloglomerular feedback mechanism, appeared to have an important role in the development of hypertension. In conclusion, experimental hydronephrosis, induced by partial ureteral obstruction, provides a new model for studies of salt-sensitive hypertension. Furthermore, the new findings imply that the current conservative treatment strategy in hydronephrosis should be reconsidered in favour of treatment that is more active, in order to prevent the development of renal injury and hypertension in later life.</p>

Numerous epidemiological studies show a strong association between low birth weight and later life hypertension and metabolic disease. Excessive in utero exposure to glucocorticoids (‘stress hormones’) has been hypothesized to be important in such developmental ‘programming’, acting via crucial physiological, gene expression or structural changes in the developing fetus. Normally, the fetus is protected from the high levels of maternal glucocorticoids by an enzymic placental barrier, 11 betahydroxysteroid dehydrogenase type 2 (11β-HSD2). In the placenta, 11β-HSD2 efficiently converts active maternal glucocorticoids (cortisol in humans; corticosterone in rodents) to physiologically inactive 11-keto forms. In previous studies in rats, maternal administration of dexamethasone, a synthetic glucocorticoid which is minimally metabolized by 11β-HSD2, or carbenoxolone, a potent inhibitor of 11 β-hydroxysteroid dehydrogenase, increased glucocorticoid load to the fetus. This resulted in lower offspring birthweight and later life hypertension and hyperglycemia — important components of the metabolic syndrome. These programming effects were seen when dexamethasone was administered selectively during the third week of gestation. We have used this well-validated model of programming to dissect the molecular mechanisms that mediate the programming of hypertension. In accord with previous observations, administration of dexamethasone (100μg/kg/day) to pregnant rats during the last week of pregnancy significantly reduced offspring birthweight by 10%. Moreover, the 9 month-old adult offspring had systolic hypertension (9% rise) accompanied by significant hypokalemia (10% fall K+). The coexistence of hypertension and hypokalemia suggested that prenatal overexposure to dexamethasone might increase mineralocorticoid activity in the kidney. Intriguingly, although offspring of dexamethasone-treated dams had 46% lower plasma renin concentrations (consistent with intravascular fluid volume expansion), 24-hour total urinary aldosterone levels were significantly reduced compared to controls (reduction of 56%). Maternal dexamethasone treatment was associated with a permanent decrease in 11β- HSD2 mRNA and activity in the kidney of the offspring (45% and 36% respectively). 11β-HSD2 plays an important role in regulation of renal sodium reabsorption (and thereby blood pressure) by acting as a pre-receptor barrier to MR access, preventing glucocorticoids from activating MR in the distal nephron. Thus, the decrease in renal 11β-HSD2 activity would allow greater endogenous glucocorticoids to activate MR, likely accounting for the low-renin, low-aldosterone hypokalemic hypertensive phenotype observed in these offspring. Other components of mineralocorticoid or glucocorticoid signaling pathways, including mineralocorticoid receptor (MR), glucocorticoid receptor (GR) and 11-beta hydroxysteroid dehydrogenase type 1 (11β-HSD1) were not altered in the offspring kidney by prenatal glucocorticoid exposure. Dexamethasone-programmed offspring also showed exaggerated mineralocorticoid activity with increased kalliuresis in response to exogenously administered corticosterone, suggesting that the decrease in renal 11β-HSD2 is functionally important. In this respect, our rat model resembles the syndrome of apparent mineralocorticoid excess where reduced 11β-HSD2 allows illicit activation of MR by glucocorticoids, resulting in excessive sodium reabsorption, hypertension and hypokalemia. We also studied the effects of maternal dexamethasone on offspring erythropoietin expression in the kidney. This followed from previous observations that identified the hepatocyte nuclear factor 4 alpha (HNF4α) as a key gene up-regulated in dexamethasone-programmed offspring liver, where it might be involved in mediating hyperglycemia. HNF4α is also expressed in the kidney. The role of HNF4α in the kidney is not fully understood, but has been implicated in regulation of erythropoietin synthesis. As in the liver, prenatal exposure to dexamethasone caused a significant increase (64% increase) in renal HNF4α expression. The increase in renal HNF4α mRNA was observed early (in one week old offspring) and persisted into adulthood. This was associated with significantly elevated levels of erythropoietin in circulation (110% increase). Moreover, animals that were exposed to prenatal dexamethasone had significantly increased red blood cell mass (7% increase), presumably as a result of upregulation of erythropoietin.

Hydronephrosis, due to ureteropelvic junction obstruction, is a common condition in infants with an incidence of approximately 0.5-1%. During the last decade, the surgical management of non-symptomatic hydronephrosis has become more conservative, and the long-term physiological consequences of this new policy are unclear. The overall aim of this thesis was to determine whether there is a link between hydronephrosis and the development of hypertension. Hydronephrosis was induced by partial ureteral obstruction in 3-week old rats or mice. In the adult animals, blood pressure was measured telemetrically during different sodium conditions and the renal function was evaluated. Both species developed salt-sensitive hypertension and histopathological changes (i.e. fibrosis, inflammation, glomerular and tubular changes) that correlated with the degree of hydronephrosis. An abnormal renal excretion pattern with increased diuresis and impaired urine concentrating ability was observed in hydronephrosis. The mechanisms were primarily located to the diseased kidney, as relief of the obstruction attenuated blood pressure and salt-sensitivity. Increased renin angiotensin system activity, due to ureteral obstruction, might be involved in the development but not necessary the maintenance of hypertension. Hydronephrotic animals displayed reduced nitric oxide availability, which might be due to increased oxidative stress in the diseased kidney. Renal nitric oxide deficiency and subsequent resetting of the tubuloglomerular feedback mechanism, appeared to have an important role in the development of hypertension. In conclusion, experimental hydronephrosis, induced by partial ureteral obstruction, provides a new model for studies of salt-sensitive hypertension. Furthermore, the new findings imply that the current conservative treatment strategy in hydronephrosis should be reconsidered in favour of treatment that is more active, in order to prevent the development of renal injury and hypertension in later life.

Hypertension and salt-sensitivity are independent risk factors for cardiovascular disease. Although both conditions are idiopathic, they develop due to a complex interplay between susceptibility genes and environmental factors. Given that the kidney plays an important role in regulating blood pressure, in particular, by maintaining sodium and water balance via pressure-natriuresis, it is not surprising that disturbances in the proper functioning of this intrarenal mechanism have been linked to these conditions. Although direct coupling of changes in renal arterial pressure (RAP) to renal interstitial hydrostatic pressure (RIHP) and consequent sodium excretion is well established, few studies have characterized the moment-to-moment aspects of this process. Thus, the main focus of the research presented herein was to characterize the moment-to-moment RAP-RIHP relationship, and assess the functioning of this intrarenal mechanism in various animal models of genetic and environmentally-induced hypertension and/or salt-sensitivity. In adult normotensive rats, the response time of RIHP to acute changes in RAP was rapid (<2 seconds), and the moment-to-moment RAP-RIHP relationship was linear over a wide range of pressures. Additionally, the functioning of this relationship was not affected by inhibition of the renin-angiotensin system and autonomic nervous system. Further, the acute RAP-RIHP relationship was impaired in hypertension and/or salt-sensitivity. Specifically, animals with a hypertensive phenotype (i.e. young spontaneously hypertensive rats [SHR] and pro-atrial natriuretic peptide gene-disrupted mice [ANP -/-]) displayed a rightward shift in the moment-to-moment pressure-natriuresis curve towards higher RAP. This rightward shift was associated with increased structurally-based vascular resistance properties in the hindlimb of young SHR versus their normotensive controls. Salt-sensitive phenotypes were associated with a blunting of this acute mechanism. Specifically, this blunting was evident in both the ANP -/-, a transgenic model of salt-sensitive hypertension, and in adult perinatal iron deficient (PID) rats, a developmentally programmed model of salt-sensitivity. It appears that a blunting in the RAP-RIHP relationship is influenced by an imbalance of key blood pressure modulating factors (e.g. ANP). Further, visceral obesity was associated with salt-sensitivity in PID rats; however the mechanism(s) are yet to be elucidated. Novel methodologies (MRI, abdominal girth) were developed for non-invasive assessment of visceral obesity to aid future research. / Thesis (Ph.D, Pharmacology & Toxicology) -- Queen's University, 2010-05-12 10:11:21.197