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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.

Cisplatin is an effective chemotherapeutic agent that is used to treat lung, breast, esophageal, ovarian, and pancreatic cancers. Despite being effective in treating a wide variety of cancer types, its cytotoxicity to off-target healthy tissues restricts its therapeutic application. The platinum atom of cisplatin interacts with DNA and results in the formation of inter-and intra-strand crosslinks, which are often referred to as DNA adducts. These adducts are mutagenic and potentially lethal to cells. The sole pathway for cells to excise intra-strand cisplatin-DNA adducts from the genome is through nucleotide excision repair. The fate of unrepaired DNA adducts is not understood fully. However, using a combination of differential centrifugation and DNA immunoblotting, we have detected cisplatin adduct-containing damaged DNA associated with small extracellular vesicles (SEVs) that are released into the cell culture medium. Moreover, the inhibition of caspase signaling blocks this release. Our observation that this response holds with multiple different cancer cell lines (U2OS, HeLa, HEK293, A375) suggests that it is a general phenomenon of cancer cell response to cisplatin. Because SEVs are involved in intercellular communication andcan transmit their contents throughout the body, this work has important implications for the systemic effects of DNA damage-based anti-cancer therapies. Moreover, the detection of cisplatin adduct-containing DNA could be useful as a marker of cancer cell killing or side effects

With a growing ageing population decline in physical function has become a major public health issue, as it is associated with disability in later life. Recent evidence suggests that blockade of the renin-angiotension-aldosterone system may have a role in improving physical function in older people.We hypothesised that inhibition of the renin-angiotensin-aldosterone system with spironolactone would improve physical function in older people without heart failure. In a double-blind, randomised controlled clinical trial 120 participants, aged >65 years with functional impairment were randomized to receive 25mg spironolactone or placebo for 20 weeks. The primary outcome was the change in six-minute walking distance over 20 weeks. Secondary outcomes were change in Timed-Get-Up and Go test, Incremental Shuttle Walk Test, measures of health related quality of life (EuroQol health questionnaire and Functional Limitation Profile) and measures of psychological state (Hospital Anxiety and Depression Scale). Outcomes measures were repeated at 10 and 20 weeks.Participant mean age was 75 years (SD 6), 65/120 (54%) were male. Only 8/120 participants (6.6%) dropped out (5 from the placebo group, 3 from the spironolactone group). Of the 112 participants who completed the study 95% (106/112) remained on medication at 20 weeks. There was no significant change in six minute walking distance at 20 weeks with a -3.2 (95% CI -28.9, 22.5) metres difference between the spironolactone group related to the placebo group (p=0.81). There was however a significant improvement in quality of life at 20 weeks (a secondary outcome) with a rise in EuroQol EQ-5D score of 0.10 (95% CI 0.03, 0.18) in the spironolactone group relative to the placebo group (p=<0.01). There were no significant changes between groups in the other secondary outcomes. This trial found that spironolactone was safe and well tolerated, but did not improve physical function in older people who did not have heart failure. Quality of life improved, but the biological plausibility and possible mechanisms for this require further study.

Abstract Aims The HOMAGE randomized trial found that spironolactone reduced left atrial volume index (LAVI), E:A ratio, and a marker of collagen type I synthesis (procollagen type I C-terminal propeptide) in patients at risk of heart failure (HF). Previous trials showed that patients with HF, preserved ejection fraction and low serum collagen type I C-terminal telopeptide to matrix metalloproteinase-1 ratio (CITP:MMP-1), associated with high collagen cross-linking, had less improvement in diastolic function with spironolactone. We evaluated the interaction between serum CITP:MMP-1 and spironolactone on cardiac function in the HOMAGE trial. Methods and results Patients at risk of HF were randomized to spironolactone (n = 260) or not (n = 255). Blood sampling and echocardiography were done at baseline, one and nine months. CITP:MMP-1 was used as an indirect measure of collagen cross-linking. Higher baseline CITP:MMP-1 (i.e. lower collagen cross-linking) was associated with greater reductions in LAVI with spironolactone at both one (p = 0.003) and nine (p = 0.01) months, but no interaction was observed for E:A ratio. Spironolactone reduced LAVI after one and nine months only for those patients in the third tertile of CITP:MMP-1 (estimated lowest collagen cross-linking) [mean differencesspiro/control: −1.77 (95% confidence interval, CI −2.94 to −0.59) and −2.52 (95% CI −4.46 to −0.58) mL/m2; interaction pacross-tertiles = 0.005; interaction pthird tertile = 0.008] with a similar trend for N-terminal pro-B-type natriuretic peptide which was consistently reduced by spironolactone only in the lowest collagen cross-linking tertile [mean differencesspiro/control: −0.47 (95% CI −0.66 to −0.28) and −0.31 (95% CI −0.59 to −0.04) ng/L; interaction pacross-tertiles = 0.09; interaction pthird tertile

Duchenne muscular dystrophy is a severe childhood-onset striated muscle disease that has no cure. The current treatment for skeletal muscle weakness has substantial side-effects. We have previously shown that treatment with drugs that inactivate the mineralocorticoid receptor (MR) improves skeletal muscle function in muscular dystrophy mice. To determine if these MR antagonists work through direct mechanisms in the skeletal muscle, we conditionally knocked out the myofiber MR in muscular dystrophy mice. Genetic ablation of MR improved skeletal muscle force and reduced fibrosis in muscular dystrophy mice similar to that observed with MR antagonist drugs. Additionally, MR antagonists stabilize fragile dystrophic skeletal muscle membranes in a MR independent manner, suggesting that these drugs have many benefits for skeletal muscle in muscular dystrophy. We then evaluated previously identified candidate MR responsive genes for their role in muscular dystrophy. None of the evaluated genes appeared to be direct myofiber specific MR targets. To investigate the role of the myofiber MR in normal muscle biology, we acutely injured the skeletal muscle of myofiber MR conditional knockout mice on a wild-type background. We found for the first time that acutely injured skeletal muscle has MR hormonal regulation and genetic ablation of the MR temporarily stabilized damaged myofibers at four days after acute muscle injury. Pharmacological inhibition of the MR with MR antagonist treatment delayed normal muscle repair in acute injury, suggesting additional roles for MR in other cell types in skeletal muscle contribute to regeneration after acute injury. These results have implications for MR modulation after acute and chronic skeletal muscle injuries.

Purpose: Cardiovascular events and complications are the major causes of death in patients with end-stage renal disease (ESRD)¹⁻³. Antihypertensive agents that block the renin-angiotensin-aldosterone system (RAAS) are considered first-line therapy in patients with ESRD as these patients have a propensity for RAAS overactivation⁴⁻⁷. Studies show that aldosterone receptor blockade reduces BP in patients with chronic kidney disease (CKD) and helps prevent negative outcomes from continued renal cellular damage⁸⁻¹⁰. Spironolactone, an aldosterone antagonist, has the potential to provoke hyperkalemia. Consequently, current guidelines do not recommend spironolactone to manage hypertension in ESRD because of this risk⁶⁻⁷. Our primary objectives were to determine the change in BP and serum potassium levels following spironolactone use. Methods: This study was a retrospective, pre-post cohort study in ESRD patients with difficult-to-control BP receiving HD. Patients prescribed spironolactone (25 mg to 50 mg) between January 2009 and January 2013 were identified using an e-prescribing record from three HD clinics in San Antonio, TX. Patients were included if they were prescribed spironolactone as 'add-on' therapy to control BP for at least 8 weeks. Results: Seventy patients were evaluated and the majority of them were overweight, diabetic, Hispanic females with a mean 65 years of age. Mean SBP and DBP decreased from baseline to week 8 [-20.74 mmHg (p < 0.0001) and -9.7 mmHg (p < 0.0426), respectively]. Mean serum potassium levels increased by an average of 0.18 mEq/L (4.5 mEq/L to 4.68 mEq/L, p = 0.09). Data analysis revealed that only 9 of 70 patients had a serum potassium level > 5.5 mEq/L at week 8. There were no adverse cardiac events reported as a result of these potassium concentrations. A two-fold decrease in SBP was seen in patients with a body mass index (BMI) > 25 kg/m² compared to patients with a BMI of ≤ 25 kg/m². At the end of the study, 23 patients (33%) achieved the goal BP for healthy adults of < 140/90 mmHg. Conclusion: These findings demonstrated that using spironolactone use in ESRD patients receiving HD can be effective and safe.

We have reported that rats subjected to partial nephrectomy (PNx) or administration of marinobufagenin (MBG) developed an increase in blood pressure, heart size, diastolic dysfunction, and cardiac fibrosis. Spironolactone (S) has been demonstrated to attenuate cardiac fibrosis in both experimental animals and humans with heart failure. We investigated the effects of treatment of S in the cardiomyopathy. Rats were divided into four groups: one group PNx and a second group sham surgery (C), placement of a minipump of S in another group and PNx + S in the final group. Rats were studied at 4 wk. PNx rats had higher systolic blood pressure, cardiac fibrosis than C. S did not differ significantly from C. PNx +. S had substantially attenuated cardiac growth and fibrosis and decreased blood pressure (all p

Monosex tilapia is desirable in aquaculture to control reproduction and produce the gender with faster growth characteristics. Phytochemicals are present in many plants and have many reported biological properties. Here we explore the use of selected phytochemicals as potential in vivo inhibitors of aromatase and as antagonist in nuclear estrogen receptors in gonad germ cells, in order to modulate the gonad differentiation process in sexually undifferentiated Nile tilapia (Oreochromis niloticus). In a first trial, diets were supplemented with genistein (500 mg/kg) and quercetin (10g/kg) along with the androgenic synthetic hormone 17á-methyltestosterone (MT) (60 mg/kg), we evaluated the in vivo response towards masculinization in genetically all-female Nile tilapia. In a second trail, experimental diets with caffeic acid (500 mg/kg), chrysin (500 mg/kg), daidzein (500 mg/kg) including MT (60 mg/kg), along with the steroidal aromatase inhibitor 1,4,6-androstatrien-3-17-dione (ATD) (150 mg/kg), and spironolactone (500 mg/kg), were administered to all-female tilapia to assess the response in final phenotypic sex of the gonad. In this trial, phytochemicals and spironolactone were also administered to all-male tilapia. A control diet, free of chemicals was included in all trials. Our results indicate that the phenotypic sex is not affected by the inclusion of phytochemicals at supplemented levels in the diet. MT and ATD induced masculinization in both feeding trials. Final male ratio with MT was 86 and 100% for experiments 1 and 2 respectively, ATD induced a 50% masculinization. Spironolactone did not affect the phenotypic gender of tilapia. Survival and growth was not different across treatments in experiment 1 and all-male fish in experiment 2. In all-female experiment 2, treatment groups for MT and ATD were significantly smaller (p