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Salt-sensitive blood pressure (BP) reflects underlying renal salt excretion impairment, suggesting compromised pressure natriuresis (PN) relationship, and vascular (endothelial) dysfunction. People with abnormal glucocorticoid homeostasis, such as in Cushing's syndrome, are often salt-sensitive but the underlying mechanisms are not clearly defined. Evidence has suggested abnormal glucocorticoid activity, via dysregulation of hypothalamic pituitary adrenocortical (HPA) axis, may have a major contributory role in salt-sensitivity. Therefore, I hypothesised that glucocorticoid excess causes vascular and renal dysfunction, contributing to salt-sensitive hypertension. I tested this hypothesis in a mouse model of ACTH-dependent Cushing's syndrome. BP was measured longitudinally in adult male C57BL/6JCrl mice by radiotelemetry, examining the effect of high salt diet (3% sodium) before and after chronic ACTH treatment. I found underlying salt-sensitivity in C57BL/6JCrl mice: BP increased by ~12 mmHg following the transition from a control salt diet (0.3% sodium) to high salt diet. Following washout, ACTH was infused by osmotic minipump, which increased daytime BP (inactive phase), flattening the diurnal BP rhythm. Reintroduction of high salt diet amplified salt-sensitivity, increasing BP ~20 mmHg. To investigate underlying mechanisms of salt-sensitivity, the acute PN relationship and vascular function were assessed in ACTH-treated mice. The acute PN relationship was unaltered with glucocorticoid excess before and after a high salt challenge. The sensitivity and maximal contractile response to vasoconstrictor phenylephrine was significantly reduced in renal arteries following glucocorticoid excess before and after a high salt challenge, with no change in mesenteric arteries. The sensitivity and maximal dilatory response to both endothelium-dependent and -independent vasodilators was reduced in renal arteries, with no changes in mesenteric arteries. Messenger RNA (mRNA) levels of glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) were assessed in renal arteries. Chronic glucocorticoid excess decreased GR but not MR mRNA in the renal artery, suggesting that glucocorticoids are primarily acting via MR in the renal artery. Therefore, I hypothesised antagonism of the MR could be protective. The effect of MR blockade (20 mg/kg/day spironolactone) on BP and vascular function following glucocorticoid excess and high salt was measured. MR blockade did not change BP but rescued the renal artery dysfunction. In other experiments, I assessed the effect of high salt diet on plasma glucocorticoid levels. High salt treatment in male C57BL/6JCrl mice increased plasma glucocorticoids. High salt also increased plasma copeptin levels, suggesting elevation of AVP. Activation of magnocellular AVP-secreting neurons could bypass glucocorticoid feedback and support sustained activation of the HPA axis. Additionally, high salt decreased hippocampal MR mRNA expression which could have implications on the tone of the HPA axis. Consistent with this, restraint test sensitivity was amplified in C57BL/6JCrl mice. In conclusion, I found a reciprocal relationship between glucocorticoids and salt. Underlying glucocorticoid excess induces renal vasodysfunction and amplifies salt-sensitive BP response. Furthermore, high salt induces activation of the HPA axis. Together, this could have long-term implications on the stress response and salt-sensitivity.

Dysregulation of the sodium-chloride cotransporter (NCC) is believed to significantly impact blood pressure. Recent studies have implicated overactivity of the sympathetic nervous system as a mechanism driving renal NCC dysregulation to evoke the development of salt-sensitive hypertension. It is proposed that the sympathetic nervous system accomplishes this by norepinephrine (NE)-mediated over-activation of the beta2-adrenergic receptors located in the distal tubules of the kidney. Beta2-adrenoreceptor activation is hypothesized to stimulate the protein kinases SPAK and OxSR1 to phosphorylate and thus activate NCC. This beta2-receptor-SPAK/OxSR1-NCC pathway was elucidated in studies that challenged salt-resistant mice with high-salt diets, bilateral adrenalectomies, and NE infusion. To expand the scope of these studies, we investigated the effects of elevated circulating NE on blood pressure, NCC activity, and expression of NCC proteins, SPAK, and OxSR1 in a different salt-resistant animal species (the Sprague-Dawley rat). In this study we implanted male Sprague-Dawley rats with osmotic minipumps delivering a subcutaneous infusion of either saline, NE, a 50:50 solution of DMSO/isotonic saline, a combination of NE and the NCC antagonist hydrochlorothiazide, or a combination of NE and the beta-adrenoreceptor antagonist propranolol. Following implantation of the pumps the rats were randomly assigned to either a standard diet (0.4% NaCl) or a high-salt diet (8% NaCl) for two weeks. After fourteen days all animals underwent acute femoral artery, vein, and bladder cannulation in order to monitor heart rate and blood pressure, administer drugs intravenously, and track renal function, respectively. Following surgical recovery, blood pressure and heart rate were measured continuously, and urine was collected in ten-minute intervals in order to assess peak natriuretic responses to amiloride and hydrochlorothiazide. Following this protocol the rats received an intravenous bolus of hexamethonium (30 mg/kg), and their peak drops in blood pressure were recorded. Afterwards both kidneys were harvested and frozen at -80 °C for measurement of NCC proteins, SPAK, and OxSR1 expression. This study demonstrates that increased circulating NE induces salt-sensitive hypertension in the naturally salt-resistant Sprague-Dawley rat. Chronic infusion of NE raised the blood pressure of the rats, and a high-salt diet exacerbated this effect. Furthermore, NE prevented salt-evoked suppression of NCC activity and NCC, SPAK, and OxSR1 protein expression. Co-infusion of hydrochlorothiazide with NE attenuated NE-mediated hypertension and caused no variance in the blood pressures between the standard salt and high-salt groups. This indicates that chronically antagonizing NCC eliminated the salt-sensitive component of NE-mediated hypertension. Beta-receptor antagonism combined with NE infusion completely eliminated the hypertensive influence of NE and downregulated the expression of NCC proteins, SPAK, and OxSR1. However, NCC activity still remained at a level comparable with that observed in the NE-infused rats, demonstrating dissociation between protein expression and function. These data, the first report in a rat model of an interaction of NE and a high salt intake that impairs NCC function, demonstrate that increased levels of NE in combination with a high dietary salt intake result in NCC dysregulation and the development of NE-mediated salt-sensitive hypertension. To an extent the data also support the proposition that NE activates the beta-adrenergic receptors to influence the activity of NCC and the expression of NCC proteins, SPAK, and OxSR1. Beta-antagonism combined with NE infusion attenuated the effects of NE on blood pressure and the expression of NCC proteins, SPAK, and OxSR1. However, the NE-mediated elevation of NCC activity still remained high. We propose that the beta-receptors are not the only adrenergic receptors that can influence NCC activity. The presence of alpha-adrenergic receptors in the distal tubules suggests that they may be able to keep NCC activity elevated through a pathway independent of the beta-receptors, SPAK, and OxSR1.