Elisa T. Marusic

Stimulation of Aldosterone Biosynthesis in Adrenal Mitochondria by Sodium Depletion

The effect of various factors on the conversion of corticosterone to aldosterone was studied in an isolated mitochondrial system from rat adrenal glands. The adrenal mitochondrial fraction from rats on a low sodium diet has a greater capacity for converting corticosterone to aldosterone than mitochondria from rats fed a normal diet. After 1 day on a low sodium diet the amount converted was 162% and after the 2nd and 4th day the amounts converted were 239 and 242%, respectively, compared to a value of 100% for the control rats. Sodium and(or) potassium added in vitro did not affect the conversion of corticosterone to aldosterone. The specificity of the sodium depletion stimulus on the conversion of corticosterone to aldosterone was established by comparing two other mitochondrial enzymes from glomerulosa cell mitochondria. Succinic dehydrogenase and 11 beta-hydroxylase were measured in normal and sodium-depleted rats and no difference in activity of either enzyme was found. The data are consistent with the view that sodium depletion stimulates the last step in aldosterone biosynthesis by causing a specific enzymatic change in adrenal mitochondria.

A Randomized, Double-Blind, Placebo-Controlled Trial of Spironolactone on Carotid Intima-Media Thickness in Nondiabetic Hemodialysis Patients

BACKGROUND AND OBJECTIVES Hemodialysis patients (HD) display high rates of cardiac diseases and mortality. In chronic kidney disease, vascular injury leads to coronary artery disease, heart failure, and stroke. Carotid intima-media thickness (CIMT) measurements are currently widely used in randomized controlled trials (RCTs) to study the efficacy of interventions. An RCT was designed for the assessment of the safety and effectiveness of spironolactone to inhibit the progression of CIMT in HD patients as a primary outcome. Secondary outcomes included measurements of plasma potassium. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS HD patients were randomly assigned to receive 50 mg spironolactone or placebo thrice weekly after dialysis. In between dialysis sessions, plasma potassium concentrations were measured every month. Ultrasonographic measurements of CIMT were done at the beginning of the study and after 2 years. RESULTS Fifty-three age- and sex-adjusted patients (30 with drug and 23 with placebo) successfully completed the trial. There were no significant differences between the two groups in all profiles studied at baseline. Measurements of CIMT after 2 years showed a progression in the placebo group, whereas in the spironolactone group a significant decrease or even reversed CIMT was observed. Progression rates (mm/yr) were: common carotid, placebo: 0.06 +/- 0.07, spironolactone: 0.01 +/- 0.04; carotid bifurcation, placebo: 0.15 +/- 0.27, spironolactone: 0.0001 +/- 0.01; internal carotid, placebo: 0.10 +/- 0.12, spironolactone: -0.10 +/- 0.15. No episodes of hyperkalemia were observed, but a slight increase in plasma potassium was found in the spironolactone group. CONCLUSIONS Fifty milligrams of spironolactone thrice weekly significantly reduced the progression of CIMT in HD patients.

Mineralocorticoid Receptor Antagonism Attenuates Cardiac Hypertrophy and Prevents Oxidative Stress in Uremic Rats

Chronic renal failure causes left ventricular hypertrophy, but the molecular mechanisms involved remain unknown. We, therefore, investigated whether the mineralocorticoid receptor is implicated in the cardiac hypertrophy observed in uremic rats and whether mineralocorticoid receptor blockade could be protective in chronic renal failure. Experimental groups were: control rats, uremic rats (NPX) with 5/6 nephrectomy (5 weeks), and NPX rats fed with spironolactone for 5 weeks. Systolic blood pressure was increased in both NPX rats and NPX rats fed with spironolactone for 5 weeks. Echocardiography revealed concentric left ventricular hypertrophy in uremia, which was attenuated by spironolactone. Enlarged cardiomyocyte size was observed in both left and right ventricles of NPX rats, an effect that was prevented by spironolactone. Mineralocorticoid receptor antagonism attenuated the increase of ventricular brain natriuretic peptide mRNA levels induced by nephrectomy. Left ventricular gene expressions of aldosterone synthase, mineralocorticoid receptor, and hydroxysteroid dehydrogenase type 2 were the same in the 3 groups, whereas gene expression of the glucocorticoid receptor was significantly diminished in chronic renal failure rats. No significant differences in cardiac aldosterone were observed between control rats and NPX rats, although NPX rats fed with spironolactone for 5 weeks showed increased plasma aldosterone levels. However, a significant increase in serum and glucocorticoid-inducible kinase-1 mRNA expression and protein was present in the NPX group; spironolactone treatment significantly reduced serum and glucocorticoid-inducible kinase-1 mRNA and protein in the left ventricle. Uremic rats exhibited a significant increase of superoxide production and reduced nicotinamide-adenine dinucleotide phosphate oxidase subunits expression (NOX-2, NOX-4, and p47phox) in the left ventricle, which was prevented by the mineralocorticoid receptor antagonist. Our findings provide evidence of the beneficial effects of spironolactone in cardiac hypertrophy and cardiac oxidative stress in chronic renal failure.

The effect of calcium on potassium-induced depolarization of adrenal glomerulosa cells

Although it has long been known that a change in membrane potential is one of the primary events involved in the transfer of information from the cell surface to the cell interior in nerve cells, it is only recently that evidence has begun to accumulate showing that changes in membrane potential also occur in a variety of hormonally responsive cell types [ 1,2]. Of particular interest to the present investigation are studies on the effects of ACTH and CAMP upon membrane potential in cells of the adrenal cortex [2], and of K+ and angiotensin II on cells of the zona glomerulosa [3]. In the zona fasciculata, ACTH causes a calcium-dependent depolarization [1,2] that is blocked by calcium channel blockers. In the zona glomerulosa, increases in extracellular [K+] lead to a calcium-dependent activation of aldosterone biosynthesis [4-61. This is associated with increased calcium uptake into the cells, and is blocked by drugs which block calcium entry [5,7]. Although it is known that large increases in extracellular [K+] induce cell membrane depolarization in nerve and muscle cells, the unique feature of the adrenal glomerulosa cells is that small changes in extracellular [K+] lead to an activation of the cell. It was, therefore, of interest to measure whether such small changes in [K+] can also lead to a depolarization of the plasma membrane in this type of adrenal cells. Here, we have studied the effect that small changes in extracellular [K+] have on the distribution of the potential-sensitive lipophilic cation, tetraphenylphosphonium [8,13], both in the presence and absence of extracellular Ca2+. The result obtained shows that small changes in extracellular [K+] lead to a calcium-dependent depolarization of the membrane.

Effect of chronic renal failure on Na,K-ATPase alpha 1 and alpha 2 mRNA transcription in rat skeletal muscle.

Previous studies have suggested that an alteration in the expression of the Na,K-ATPase of muscle may be an important determinant of enhanced insulin sensitivity in chronic renal failure. Therefore, in the present studies we have examined the effect of uremia on the Na,K-ATPase alpha isoforms in skeletal muscle, at the level of mRNA expression and enzymatic activity. The activity of the sodium pump, as measured ouabain-sensitive 86Rb/K uptake in soleus muscle, revealed a reduction in the activity in uremia, related to the increment in plasma creatinine values. The decrement in 86Rb uptake by the rat soleus muscle of experimental animals was associated with changes on Na,K-ATPase gene product. Northern analysis of mRNA revealed isoform-specific regulation of Na,K-ATPase by uremia in skeletal muscle: a decrease of approximately 50% in alpha 1 subunit Na,K-ATPase mRNA, as compared to controls. The decrement in alpha 1 mRNA correlates with the decreased activity of the Na,K-ATPase in uremia, under basal conditions and with the almost complete inhibition of the Na,K-ATPase, of uremic tissue by a concentration of 10(-5) M ouabain. Although the activity of the alpha 2 isoform pump was not modified by uremia, the 3.4-kb message for this enzyme was increased 2.2-fold; this discrepancy is discussed. Altogether these findings demonstrate that the defective extrarenal potassium handling in uremia is at least dependent in the expression of alpha 1 subunit of the Na,K-ATPase.

Effect of a Simultaneous Potassium and Carbohydrate Load on Extrarenal K Homeostasis in End-Stage Renal Failure

Patients with chronic renal failure (CRF) are continuously exposed to hyperkalemia. In these patients the extrarenal disposal of a potassium load may be very important to determine the plasma potassium levels. We studied the effect of a combined oral load of potassium (0.5 mEq/kg body weight) and carbohydrate (0.5 g/kg body weight) to mimic normal ingestion of potassium. Eight CRF patients and 5 control subjects were studied. The maximal increase in plasma potassium levels achieved was significantly higher in the patients (1.07 +/- 0.1 mEq/l) than in controls (0.39 +/- 0.05 mEq/l). Basal insulin levels were higher in the CRF patients and increased with the oral potassium and carbohydrate load in both controls and patients. In the CRF patients only 58.9 +/- 3% of the potassium load was translocated to the intracellular space compared to 81 +/- 6% in the controls. No correlation was found between the acid base status and maximal potassium increase. We conclude that patients with CRF exhibit an impaired extrarenal handling of potassium and that this abnormality does not appear to be related to insulin secretion or acid base status.

Adrenal-dependent modulation of the catalytic subunit isoforms of the Na+-K+-ATPase in aorta

Na+-K+-ATPase gene expression and activity were studied in aortas from adrenalectomized (ADX) rats and ADX rats with deoxycorticosterone supplement (ADX-DOCA). Northern analysis of RNA from ADX rats revealed a significant decrease in α2-mRNA levels (38.5 ± 8.3% of control, P < 0.01) that was prevented by DOCA ( P < 0.05). A decrease to 55.8 ± 7.7% in α2-isoform protein was observed 8 days after adrenal removal ( P < 0.05); DOCA reversed this effect (90.8 ± 10.5%). Adrenalectomy induced a decrease of 68.5 ± 4.5% in β1-mRNA ( P < 0.01) and 52.7 ± 8.3% in ADX-DOCA rats ( P < 0.01). Also, a reduction in β1-isoform protein that was not prevented by DOCA was detected after adrenalectomy (47.1 ± 11%, P < 0.01). In contrast, no differences in α1-mRNA or -protein levels were observed. Vascular sodium pump activity was reduced to 59.8 ± 4.6% of control values after adrenalectomy ( P < 0.01); this reduction was reversed by DOCA. Our data indicate that corticosteroids regulate Na+-K+-ATPase isoform expression and activity in vascular tissue in vivo, suggesting a mineralocorticoid-dependent modulation of α2-Na+-K+-ATPase gene expression in aorta, with β1-isoform expression dependent on the presence of glucocorticoids.Na+-K+-ATPase gene expression and activity were studied in aortas from adrenalectomized (ADX) rats and ADX rats with deoxycorticosterone supplement (ADX-DOCA). Northern analysis of RNA from ADX rats revealed a significant decrease in alpha2-mRNA levels (38.5 +/- 8.3% of control, P < 0.01) that was prevented by DOCA (P < 0.05). A decrease to 55.8 +/- 7.7% in alpha2-isoform protein was observed 8 days after adrenal removal (P < 0.05); DOCA reversed this effect (90.8 +/- 10.5%). Adrenalectomy induced a decrease of 68.5 +/- 4.5% in beta1-mRNA (P < 0.01) and 52.7 +/- 8.3% in ADX-DOCA rats (P < 0.01). Also, a reduction in beta1-isoform protein that was not prevented by DOCA was detected after adrenalectomy (47.1 +/- 11%, P < 0.01). In contrast, no differences in alpha1-mRNA or -protein levels were observed. Vascular sodium pump activity was reduced to 59.8 +/- 4.6% of control values after adrenalectomy (P < 0.01); this reduction was reversed by DOCA. Our data indicate that corticosteroids regulate Na+-K+-ATPase isoform expression and activity in vascular tissue in vivo, suggesting a mineralocorticoid-dependent modulation of alpha2-Na+-K+-ATPase gene expression in aorta, with beta1-isoform expression dependent on the presence of glucocorticoids.

Studies on the Stannius corpuscles of the Chilean clingfish, Sicyases sanguineus

Abstract Vargas and Concha [Vargas F., F., and Concha B., J. (1957), Invest. Zool. Chil. 3, 81–87; 88–95] described some encapsulated glands in the posterior part of the kidneys of the Chilean clingfish, Sicyases sanguineus , and called them the interrenals. Present investigations showed that the surgical removal of these glands consistently produced hypercalcemia. No significant effects were seen in plasma sodium, potassium, and magnesium levels and in percentage hematocrit and body weight change, as was once reported previously in killifish [Pang, P. K. T., Pang, R. K., and Griffith, R. W. (1975). Gen. Comp. Endocrinol. 26, 179–185]. We conclude that these glands are the Stannius corpuscles and not the interrenals of this fish. Histological studies showed the high vascularization and the follicular arrangement of cells.

Osmotic adaptations of the Chilean clingfish, Sicyases sanguineus, during emertion

Abstract 1. 1. The osmotic adaptations for dehydration of the Chilean clingfish ( Sicyases sanguineus ) were examined in emerged fish. 2. 2. Plasma osmolarity, electrolyte levels, body weight changes, hematocrit, muscle water content and urea were measured at various times up to 24 hr of emersion. 3. 3. At 24 hr of emersion, the gut surface of the fish was reddish with engorged capillaries. The concomitant presence of air in the gut suggests a role for the intestine in the air breathing mechanism. 4. 4. S. sanguineus maintained out of water showed a significant loss of body weight and increases in hematocrit values and plasma osmolarity, urea and electrolyte levels.

An unusual pattern of Na+ and K+ movements across the horse erythrocyte membrane

Marked differences in the activities of three monovalent cation transport systems in horse versus human erythrocytes are reported. Whereas horse erythrocytes exhibit a 6-fold higher sodium-lithium countertransport, the unidirectional flux of potassium through the sodium pump is 3-4 times slower and the sodium-potassium cotransport system cannot be detected. In spite of this, horse and human cells are able to maintain similar Na+ and K+ gradients.