low-Na Diet Research Articles

Altered potassium balance and aldosterone secretion in a mouse model of human congenital long QT syndrome.

The voltage-dependent K(+) channel responsible for the slowly activating delayed K(+) current I(Ks) is composed of pore-forming KCNQ1 and regulatory KCNE1 subunits, which are mutated in familial forms of cardiac long QT syndrome. Because KCNQ1 and KCNE1 genes also are expressed in epithelial tissues, such as the kidneys and the intestine, we have investigated the adaptation of KCNE1-deficient mice to different K(+) and Na(+) intakes. On a normal K(+) diet, homozygous kcne1(-/-) mice exhibit signs of chronic volume depletion associated with fecal Na(+) and K(+) wasting and have lower plasma K(+) concentration and higher levels of aldosterone than wild-type mice. Although plasma aldosterone can be suppressed by low K(+) diets or stimulated by low Na(+) diets, a high K(+) diet provokes a tremendous increase of plasma aldosterone levels in kcne1(-/-) mice as compared with wild-type mice (7.1-fold vs. 1.8-fold) despite lower plasma K(+) in kcne1(-/-) mice. This exacerbated aldosterone production in kcne1(-/-) mice is accompanied by an abnormally high plasma renin concentration, which could partly explain the hyperaldosteronism. In addition, we found that KCNE1 and KCNQ1 mRNAs are expressed in the zona glomerulosa of adrenal glands where I(Ks) may directly participate in the control of aldosterone production by plasma K(+). These results, which show that KCNE1 and I(Ks) are involved in K(+) homeostasis, might have important implications for patients with I(Ks)-related long QT syndrome, because hypokalemia is a well known risk factor for the occurrence of torsades de pointes ventricular arrhythmia.

Activation of epithelial Na channels during short-term Na deprivation.

The role of epithelial Na channels in the response of the kidney to short-term Na deprivation was studied in rats. Animals were fed either a control-Na (3.9 g/kg) or a low-Na ( 3.8 mg/kg) diet for 15 h. Urinary excretion of Na (micromol/min), measured in conscious animals in metabolic cages, was 0.45 +/- 0.07 in controls and 0.04 +/- 0.01 in Na-deprived animals. Glomerular filtration rate, measured as the clearance of creatinine, was unaffected by the change in diet, suggesting that the reduced Na excretion was the result of increased Na reabsorption. K excretion (micromol/min), increased after the 15-h period of Na deprivation from 0.70 +/- 0.10 to 1.86 +/- 0.19. Thus the decrease in urine Na was compensated for, in terms of electrical charge balance, by an increase in urine K. Plasma aldosterone increased from 0.50 +/- 0.08 to 1.22 +/- 0.22 nM. Principal cells from cortical collecting tubules isolated from the animals were studied by using the patch-clamp technique. Whole cell amiloride-sensitive currents were negligible in the control group (5 +/- 4 pA/cell) but substantial in the Na-deprived group (140 +/- 28 pA/cell). The abundance of the epithelial Na channel subunits, alpha, beta, and gamma in the kidney was estimated by using immunoblots. There was no change in the overall abundance of any of the subunits after the 15-h Na deprivation. However, the apparent molecular mass of a fraction of the gamma-subunits decreased as was previously reported for long-term Na deprivation. Calculations of the rate of Na transport mediated by the Na channels indicated that activation of the channels during short-term Na deprivation could account in large part for the increased Na reabsorption under these conditions.

A High Salt Diet Increases Renal Nitrotyrosine Formation and Urinary Excretion of F 2 -Isoprostanes and Protein in Dahl Salt-Sensitive Rats

P28 Superoxide formation causes tissue injury in many disease processes, and interaction between superoxide and nitric oxide forms peroxynitrite, a very strong oxidant. An index of peroxynitrite damage to tissues is nitrotyrosine formation. Also, F 2 -isoprostanes have been shown to be a reliable marker of oxidative stress in vivo. Recently, oxidative stress was shown to contribute to the maintenance of hypertension in the SHR; however, whether oxidative stress is involved in the renal damage that occurs in Dahl salt-sensitive (S) hypertension is not clear. Our goal was to determine the role of oxidative stress in the renal damage that occurs in Dahl S/Rapp rats on high Na intake by determining the urinary excretion of F 2 -isoprostanes and renal nitrotyrosine formation and determining urinary protein excretion as an index of renal damage. Studies were conducted in S rats on a low salt (0.3% NaCl) or a high salt (8% NaCl) diet for 3 weeks. After 3 weeks urine was collected continuously for 24 hours, and urinary F 2 -isoprostane concentration was measured with a gas chromatography/mass spectrometric assay. Renal nitrotyrosine formation was determined by immunohistochemistry and urinary protein excretion by the Bradford method. We found that high Na intake significantly increased urinary F 2 -isoprostane excretion to 24.2±1.7 ng/day compared with 9.7±0.8 ng/day in rats on a low Na diet, P<0.05. Renal glomerular nitrotyrosine was prevalent in the high Na group compared to the low Na group. Urinary protein excretion was 274±32 mg/day in high Na rats and 55±11 mg/day in low Na rats, P<0.05. The results suggest that oxidative stress is significantly increased in Dahl S rats on a high salt diet, which possibly contributes to the progression of renal damage.

Endothelin-B Receptors Play an Important Role in Modulating Chronic Pressure-Natriuresis and Blood Pressure Regulation in Response to Changes in Dietary Sodium Intake.

25 Activation of the ET B receptor by endothelin (ET-1) has been shown to inhibit renal tubule transport, dilate renal microcirculatory vessels, and inhibit renin release. The purpose of this study was to determine the role of ET and ET B receptors in modulating renal-pressure natriuresis and blood pressure regulation in response to changes in dietary Na intake. To test whether the renal production of ET is enhanced in response to elevation in Na intake, the effects of a low (0.4%), normal (1.0%), or high (8%) Na diet on 24hr urinary excretion of ET-1 and mRNA expression of preproendothelin (ribonuclease protection assay) were determined. Increasing Na intake from low to normal levels had no effect on urinary ET-1 excretion or renal expression of preproendothelin. In contrast, increasing Na intake from normal to high levels resulted in significant increases in urinary excretion of ET-1 (7223±968 vs 2127±167 pg/24hrs) and expression of preproendothelin (42.3±6.7 vs 25.4±1.8 densitometric units) in the renal medulla. To test whether ET B receptors play an important role in modulating arterial pressure in response to changes in Na intake, the relationship between arterial pressure and sodium excretion was determined in control rats and in ETB receptor antagonist-treated rats (A-192621, 30mg/kg/day for 7 days). Long-term ET B receptor blockade resulted in a significant rightward shift in the chronic pressure-natriuresis relationship. In control rats on a normal and high Na intake, AP was 122±3 and 132±3mmHg, respectively. In contrast, AP in ETB receptor antagonist-treated rats on a normal and high Na intake was 144±2 and 171±12mmHg, respectively. In summary, we report that the renal endothelin system is upregulated in response to increases in sodium intake. Blockade of the ET B receptors results in significant hypertension that is salt-sensitive. These results indicate that endothelin via ET B receptors plays an important role in modulating renal-pressure natriuresis and blood pressure regulation in response to changes in dietary Na intake.

Hydrolysis and absorption in the small intestines of posthatch chicks

In the immediate posthatch period, chicks must transfer from metabolic dependence on yolk to utilization of exogenous feed. This study describes changes in intestinal luminal pancreatic enzyme activity and mucosal uptake posthatch as influenced by feed and Na intake. Chicks with access to feed increased in BW and small intestinal weight in the 48-h posthatch, whereas chicks without access to feed decreased in BW; however, small intestinal weight increased during this period. Chicks ingesting feed showed increases in total intestinal trypsin, amylase and lipase activities that were correlated with intestinal weights and BW. Chicks without access to feed showed little change in trypsin and amylase activities, and these increased only after feed consumption. Feeding a low-Na diet did not significantly change the regression coefficient between pancreatic enzyme activity and BW. Mucosal uptake was estimated by measuring Na+,K+-adenosine triphosphatase (ATPase) activity in small intestinal segments. In fed birds this activity increased in relationship to growth, whereas in nonfed birds uptake increased only after access to feed. Low-Na diets allowed only minimal mucosal uptake in all intestinal segments. This study indicates that secretion of trypsin and amylase into the intestine was triggered by feed intake. In addition, Na plays a critical role in intestinal uptake in the immediate posthatch period.

Differential subcellular localization of ENaC subunits in mouse kidney in response to high- and low-Na diets.

Previous electrophysiological experiments on renal cortical collecting ducts indicated that dietary sodium intake and variations in aldosterone plasma levels regulate the abundance of functional epithelial Na channels (ENaC) in the apical plasma membrane. In mouse kidney we investigated by immunohistochemistry whether feeding for 3 wk a diet with high (3% Na) and low (0.05% Na) Na content influences the distribution pattern of ENaC. In mice of all experimental groups, ENaC was apparent in cells from the late portion of the distal convoluted tubule (DCT2) down to the medullary collecting duct (CD). In mice on a high-Na diet (plasma aldosterone: 40.8 +/- 2.0 ng/dl), the alpha-subunit was undetectable, and the beta- and gamma-ENaC were detected in the cytoplasm, but not in the apical plasma membrane of the cells. In contrast, in mice on a low-Na diet (plasma aldosterone: 93.6 +/- 9.3 ng/dl) all three ENaC subunits were displayed in the subapical cytoplasm and in the apical membrane of DCT2, connecting tubule (CNT), and, although less prominent, in cortical CD cells. Apical plasma membrane immunostaining progressively decreased along the cortical CD, simultaneously with increasing cytoplasmic staining for beta- and gamma-ENaC. Thus our data on mice adapted to moderately low and high Na intake suggest that regulation of ENaC function in vivo involves shifts of beta- and gamma-subunits from the cytoplasm to the apical plasma membrane and vice versa, respectively. The insertion of these subunits into the apical plasma membrane coincides with upregulation of the alpha-subunit and its insertion into the apical plasma membrane.

Sodium pump inhibition and regional expression of sodium pump alpha-isoforms in lens.

Both hypertension and cataract formation have been associated with reductions in sodium pump activity, possibly as a result of an endogenous inhibitor. The objective of the present study was to answer 4 closely related questions: (1) Is the lens sodium pump effectively inhibited by a labile, digitalis-like factor we have identified in the peritoneal dialysate from hypertensive patients in end-stage renal failure? (2) How does that inhibition compare to that induced by ouabain? (3) Does sodium pump isoform distribution determine the degree of lens sodium pump inhibition? (This question was precipitated by the unanticipated finding that the labile DLF was more effective in inhibiting lens sodium pump than was anticipated.) (4) Is sodium pump activity altered in lens in response to increased salt intake, a maneuver known to increase endogenous digitalis-like factor? We found that whereas ouabain produced equivalent or significantly less inhibition of lens Na(+), K(+)-ATPase from calf or rabbit, respectively, compared with brain, labile digitalis-like factor preferentially inhibited lens compared with brain. Analysis of whole-lens preparations from rabbit, calf, and normal human lens revealed substantial alpha2- and alpha3-isoforms of the sodium pump but little alpha1-isoform. Ouabain inhibition of whole-lens Na(+),K(+)-ATPase from rabbit and calf were comparable: for rabbit lens, K(i)=5.2x10(-7) mol/L; for calf lens, K(i)=1.0x10(-6) mol/L. Limited quantities of labile digitalis-like factor prohibited similar determinations; however, its concentration-activity profile paralleled that of ouabain. Na(+), K(+)-ATPase activity, measured in the 3 major anatomic regions of lens and normalized to nucleus, was greatest in epithelium (56. 9+/-17.9) compared with cortex (5.8+/-1.4) and nucleus (1.0+/-0.0; P=0.01). Immunohistochemistry of rabbit lens found abundant alpha2- and alpha3-isoforms in epithelium and limited alpha3 but undetectable alpha1 in cortex and nucleus. Finally, rats randomized to a high Na diet showed significantly reduced lens Na(+), K(+)-ATPase activity compared with those on a low Na diet, consistent with the effects of a sodium pump inhibitor. In conclusion, the present study suggests that digitalis-like factor may provide a link between hypertension and cataract formation.

An experimental rat model of salt-sensitive hypertension; biochemical and morphological parameters and sympathetic nervous system.

The objective of the study was to outline the characteristics of the development of hypertension and some neurohumoral, haematological and morphological factors contributing to development of high blood pressure in a genetic model of salt-sensitive rat. Characteristics of Dahl salt-sensitive (DS) rats, as compared to their Dahl salt-resistant (DR) controls were as follows: 1) DS rats display higher blood pressure and lower heart rate compared to DR rats as early as 1 month of age at weaning). They gradually develop hypertension at 2 months of age, irrespective of diet. Low-Na diet (0.5% NaCl) does not prevent hypertension but delays its development and ameliorates it. High Na-diet (8% NaCl) exacerbates hypertension. 2) DS rats have retardation in body weight gain. They develop mild hypochromic anaemia. 3) After 2 months of Na loading (3 months of age), DS rats express significantly increased Na and water retention and increased plasma volume by 15% compared to 2.8% increase in DR rats on high-Na diet. 4) DS rats showed renal parenchymal lesions, more pronounced after Na-loading, focal atrophy of cortical tubules, mesangial matrix expansion and glomerulosclerosis. Consistent with high blood pressure were changes in renal arterioles, fibromuscular proliferation, deposition of fibrinoid material in intima. 5) Sodium loading produced increased activity of the sympathetic nervous system (SNS), and sodium restriction reduced SNS responsiveness.

Stress and salt intake: experimental data on Dahl salt­resistant and salt­sensitive rats

The objective of the study was to evaluate the susceptibility of genetically predisposed to salt sensitivity Dahl rats to psychological stress and the effect of sodium ion on stress development. After weaning, Dahl salt-resistant (DR) and two groups of salt-sensitive (DS) rats were fed low-Na (0.5 per cent NaCI) and high-Na (8 per cent NaCI) diets for 2 months. A constellation of sensorimotor and behavioural tests were employed. The psychological stress was produced using a new model of acoustic startle device. DS weanling rats showed significantly increased sensorimotor reactivity, increased emotionality and increased sensitivity to acoustic stress, as compared to DR weanling rats. All the above parameters were exaggerated after 2-month Na loading: there was a significant decrease in the latency of the stressful response and increased magnitude of the hypertensive response to auditory stress. Blood pressure of Na-loaded DS rats increased by 43 per cent after auditory stress, as compared to 18 per cent increase in the Na-restricted rats. The effect of Na ion was mediated by an increased activity of the sympathetic nervous system (SNS). The chronic effect of Na restriction showed reduced SNS responsiveness and diminished neurohumoral reserve to maintain cardiovascular homeostasis. Risks of both Na loading and Na depletion in sodium sensitivity were discussed.

Regulation of renal alpha2-adrenoceptor activity in Dahl salt-sensitive rats by dietary sodium changes

The objective of the study was to evaluate the concept that genetically predisposed salt-sensitivity contributes to an increased adrenergic susceptibility and renal alpha 2-adrenoceptor (A2) abnormality in Dahl salt-sensitive hypertensive rats. The results showed: i) After 2 months of Na-loading (8% NaCl) Dahl salt-sensitive (DSS) rats expressed increased sodium and water retention which paralleled gradual development of diastolic hypertension. Low Na diet (0.5% NaCl) does not prevent hypertension but delays its development. ii) The increased activity of the sympathetic nervous system (SNS), in DSS rats corresponded to the development of hypertension and was stimulated by Na-loading. It was assessed by plasma catecholamine levels and heart rate changes. iii) The increased density of renal A2 by 29% was upregulated by high sodium diet, and coupled with increased norepinephrine level by 53%, only in DSS but not in DSR rats. iv) No strain renal A2 and epinephrine differences between DSS and DSR were found in weanling, prehypertensive rats, or in the adult DSS and DSR on low Na diet. By mediating an enhanced receptor-coupled response, such as increased proximal tubular sodium reabsorption during sodium loading, a genetic abnormality of renal alpha 2-adrenoceptors may contribute to some of the pathophysiologic derangements leading to hypertension in Dahl salt-sensitive rats.

Sympathetic and platelet adrenergic activity and salt sensitivity: an experimental study.

The objective of the study was to evaluate the advanced hypothesis that genetically predisposed salt-sensitivity contributes to an increased adrenergic susceptibility and platelet activity and both these factors play a role in the pathogenesis of hypertension in a Dahl rat model. The results showed: i) Dahl salt-sensitive rats (DS) gradually develop a diastolic hypertension by the end of 3 months of age, in spite of the diet they are fed. Low-Na diet (0.5% NaCl) does not prevent hypertension but delays its development. High-Na diet (8%) exacerbates their hypertension. ii) After 2 months of Na-loading, DS rats expressed significantly increased sodium and water retention and increased plasma volume by 15%, compared with 2.8% in Dahl salt-resistant (DR) rats on high-Na diet. iii) The increased activity of the sympathetic nervous system (SNS) in DS rats paralleled the development of hypertension and was stimulated by Na-loading. It was assessed by their catecholamines status and heart rate changes. iv) Platelet activity of DS rats was increased as reflected in collagen-induced nonstimulated and adrenaline-stimulated aggregation, and an increased plasma T x B2/6-keto PGF1 alpha ratio. Na-loading further increased platelet activity. v) Both DR and DS rat platelets displayed alpha 2-adrenoceptors (A2) of low binding capacity (Bmax 25 and 35 fmol/mg protein, respectively) and low affinity (KD 5.6 nM for both groups), suggesting that platelet alpha 2 adrenoceptors in this strain of rats might not play a significant biological role in their increased platelet activity. The fact that platelet alpha 2-adrenoceptors do not define the stimulation of SNS in DS rat do not exclude their participation in development of salt-dependent hypertension, since a genetic defect(s) of these ubiquitous receptors (brain, kidney, blood vessels) could still contribute to the pathogenesis of the disease.

Solute composition and heat shock proteins in rat renal medulla.

The high content of heat shock proteins (HSPs) 25 and 72 in the hyperosmotic inner medulla of the concentrating kidney has been ascribed to the high NaCl and urea concentrations in this kidney zone. To assess the effects of variations in the composition of solutes in the renal medulla on the intrarenal distribution of HSPs, rats were fed either a high- or low-Na diet for 3 weeks. These diets result in greatly differing urine and inner medullary solute composition. Sodium dodecyl sulphate polyacrylamide gel electrophoresis and Western blot techniques were used to analyse HSP25 and HSP72 in the cortex, outer medulla and inner medulla. In addition, the amounts of organic osmolytes (sorbitol, myo-inositol, betaine and glycerophosphorylcholine) and urea in the tissue were determined by high-performance liquid chromatography. Intra- and extracellular electrolyte concentrations at the papillary tip were measured by electron microprobe analysis. In the high-Na group, urine osmolality was about 1000 mosmol/kg lower than in rats fed a low-Na diet, due to lower urea concentrations. The sum of urine sodium and potassium concentrations, however, did not differ between the two groups. Neither in the outer nor in the inner medulla was the sum of the concentrations of organic osmolytes affected by the dietary treatment. The sum of sodium, potassium and chloride concentrations did not differ between the two experimental groups, neither in the interstitial nor in the intracellular compartments. However, the urea content and the amounts of HSP25 and HSP72 were significantly lower in the inner medulla of the group of rats fed a high-Na diet. Our results suggest that urea participates in the regulation of the medullary levels of the HSPs and that both HSP25 and HSP72 are components of mechanisms protecting medullary cells against the deleterious effects of high urea concentrations.

Evaluation of a rapid protocol for the assessment of salt sensitivity against the blood pressure response to dietary sodium chloride restriction.

The "gold standard" for the assessment of salt sensitivity of hypertension is the blood pressure response to dietary NaCl restriction; nevertheless, for practical purposes, a more rapid test that would not depend on the patient's compliance to the dietary prescription would be very useful in clinical research and medical practice. The aim of this study was thus to evaluate the effectiveness and reliability of a rapid, easy-to-standardize protocol for the assessment of salt sensitivity against the blood pressure response to dietary salt restriction. A total of 108 hypertensive patients were screened for salt sensitivity by the modified protocol of Grim et al. Thereafter, nine patients identified by the test as salt sensitive and nine identified as salt resistant followed, for two consecutive periods of 1 week, a diet with normal (200 mmol/day) or low (50 mmol/day) NaCl content. Compliance to the diet was checked by repeated 24-h urine collections. The group as a whole experienced a significant fall in blood pressure during the low Na diet (mean pressure = 123 +/- 3 v 118 +/- 3 mm Hg; P < .05). However, whereas patients identified as salt sensitive by the Grim protocol had a marked and significant blood pressure decrease (systolic -12 mm Hg, diastolic -7 mm Hg), no change was observed in those classified as salt resistant (systolic -2 mm Hg, diastolic -2 mm Hg). A significant correlation between changes in urinary Na excretion and changes in blood pressure was found only in salt-sensitive hypertensive patients. In conclusion, the modified Grim protocol tested in this study was able to correctly predict a significant blood pressure response to dietary salt restriction in the majority of cases. A validation of this test in a larger patient population may be advisable.

Evaluation of a rapid protocol for the assessment of salt sensitivity against the blood pressure response to dietary sodium chloride restriction

The “gold standard” for the assessment of salt sensitivity of hypertension is the blood pressure response to dietary NaCl restriction; nevertheless, for practical purposes, a more rapid test that would not depend on the patient's compliance to the dietary prescription would be very useful in clinical research and medical practice. The aim of this study was thus to evaluate the effectiveness and reliability of a rapid, easy-to-standardize protocol for the assessment of salt sensitivity against the blood pressure response to dietary salt restriction. A total of 108 hypertensive patients were screened for salt sensitivity by the modified protocol of Grim et al. Thereafter, nine patients identified by the test as salt sensitive and nine identified as salt resistant followed, for two consecutive periods of 1 week, a diet with normal (200 mmol/day) or low (50 mmol/day) NaCl content. Compliance to the diet was checked by repeated 24-h urine collections. The group as a whole experienced a significant fall in blood pressure during the low Na diet (mean pressure = 123 ± 3 ν 118 ± 3 mm Hg; P < .05). However, whereas patients identified as salt sensitive by the Grim protocol had a marked and significant blood pressure decrease (systolic −12 mm Hg, diastolic −7 mm Hg), no change was observed in those classified as salt resistant (systolic −2 mm Hg, diastolic −2 mm Hg). A significant correlation between changes in urinary Na excretion and changes in blood pressure was found only in salt-sensitive hypertensive patients. In conclusion, the modified Grim protocol tested in this study was able to correctly predict a significant blood pressure response to dietary salt restriction in the majority of cases. A validation of this test in a larger patient population may be advisable.

Angiotensin-independent mechanism for aldosterone synthesis during chronic extracellular fluid volume depletion.

Wild-type (Agt+/+) and homozygous angiotensinogen deletion mutant (Agt-/-) littermates were placed on normal (NS) or low Na diet (LS) for 2 weeks. Plasma aldosterone levels (P(aldo)) were comparable during NS, and similarly elevated during LS in Agt+/+ and Agt-/-. Moreover, in both, the elevation in P(aldo) was accompanied by marked increase in adrenal zona glomerulosa cells and adrenal P450aldo mRNA. Agt-/- mice were distinguished from Agt+/+ mice by their higher plasma K level, by approximately 1.5 and approximately 3.8 mEq/liter during NS and LS, respectively. Within the Agt-/- group, P(aldo) was directly proportional to plasma K. The importance of K for the hyperaldosteronism during dietary Na restriction was verified by the observation that superimposition of K restriction led to hypotension in Agt+/+ and uniform death in Agt-/- mice along with a reduction in P(aldo) by 75 and 90%, respectively. Thus, suppression of potassium, but not angiotensin, led to a marked attenuation of hyperaldosteronism during dietary Na restriction. Therefore, (a) a powerful angiotensin-independent mechanism exists for the hyperaldosteronism during LS; (b) high K is a central component of this mechanism; (c) contrary to current belief, the tonic effect of high K on aldosterone synthesis and release does not require an intact renin-angiotensin system; and (d) normally, intermediary feedback signals for hyperaldosteronism, i.e., both hypotension and high K, are effectively masked by aldosterone actions.

Regulation of aldosterone synthase cytochrome P450 (CYP11B2) and 11 beta-hydroxylase cytochrome P450 (CYP11B1) expression in rat adrenal zona glomerulosa cells by low sodium diet and angiotensin II receptor antagonists.

Changes in the mRNA levels for aldosterone synthase cytochrome P450 (CYP11B2 or P450aldo) and 11 beta hydroxylase cytochrome P450 (CYP11B1 or P45011 beta) in rat adrenal glands were studied in response to angiotensin II type 1 (AT1) and type 2 (AT2) receptor antagonists. CYP11B1 and CYP11B2 genes were highly homologous (88.5%) in their nucleotide sequences of the amino acid coding regions. Reverse transcription-polymerase chain reactions (RT-PCR) which are capable of discriminating between rat CYP11B1 and CYP11B2, were performed with specific primers for each P450. Upon sodium restriction (5 mmol Na(+)/kg of diet) of rats for 14d, the amount of the CYP11B2 mRNA in the adrenal glands was increased 8.5-fold compared to that from the rats fed a normal diet (225 mmol Na(+)/kg of diet), whereas no significant change in the CYP11B1 mRNA was observed after the dietary sodium restriction. As shown by an immunoblot analysis, the adrenal capsule portions (mainly zona glomerulosa) of the rats kept on the low Na diet for 14d expressed significantly higher levels of both CYP11B2 and CYP11B1, and contained a significantly higher amount of CYP11B2 than those from the rats fed by normal diet. The activities of the CYP11B2 enzyme were also found to be increased by about 8-fold on day 14. In concert with these alterations, the plasma aldosterone concentration (PAC) increased. However, when the specific AT1 antagonist E4177 was given to rats maintained on the low Na diet, the amount and activity of CYP11B2, as well as the PAC, were suppressed. In contrast, the increase in CYP11B2 induced by the low Na diet was not affected by the AT2-specific antagonist PD123177. These results indicate that the aldosterone synthase cytochrome P450 (CYP11B2) is an ultimate target of the regulation of aldosterone biosynthesis by an AT1 receptor antagonist.

Expression of aldosterone synthase cytochrome P450 (P450aldo) mRNA in rat adrenal glomerulosa cells by angiotensin ii type1 receptor

Changes in the mRNA levels for aldosterone synthase cytochrome P450 (P450aldo or CYP11B2) in rat adrenal glands were studied in response to angiotensin II type 1 (AT1) and type 2 (AT2) receptor antagonists. Since 11β hydroxylase P450 (P45011β or CYP11B1), which shows high homology (88.5%) with P450aldo in their nucleotide sequences of the amino acid coding regions, is also expressed in the adrenal gland, RT-PCR was performed with specific primers for each P450. Upon sodium restriction (5 mmol Na+ /kg of diet) of the rats for 14 days, the amount of the P450aldo mRNA in the adrenal glands increased 8.5 fold above from the rats fed on a normal diet (225 mmol Na+ /kg of diet), whereas no significant change of the P45011β mRNA was observed after the dietary sodium restriction. As shown by an immunoblot analysis, the adrenal capsule portions (mainly zona glomerulosa) of the rats kept on the low Na diet for 14 days expressed significantly higher level of P450aldo than those from the rats fed the normal diet. In concert with the alteration, plasma aldosterone concentration increased. However, when a specific AT1 antagonist E4177 was given to the rats kept on a low Na diet, the amount and activity of P450aldo as well as the plasma aldosterone concentration was suppressed. On the other hand, the increase of P450aldo induced by the low Na diet was not affected by an AT2-specific antagonist, PD123177.

Regulation of Na channels in the rat cortical collecting tubule: effects of cAMP and methyl donors.

The whole cell patch-clamp technique was used to investigate the interactions of the amiloride-sensitive Na channel of the rat cortical collecting tubule (CCT) with adenosine 3',5'-cyclic monophosphate (cAMP) and with methyl donors. The amiloride-sensitive whole cell current (INa) was measured in principal cells of dissected, split-open tubules from rats maintained either on a control diet or on a low-Na diet to increase endogenous aldosterone secretion. With Na-depleted animals, INa was highest immediately after rupture of the membrane patch and averaged 325 pA at a membrane potential of -60 mV. INa declined over 15 min to approximately 35% of the initial value. With 8-(4-chlorophenylthio)-cAMP in the pipette, INa increased within 5 min of membrane rupture and was maintained for 15 min at levels three- to fourfold higher than the corresponding control values. With Na-replete animals, INa was undetectable (< 10 pA) without cAMP. With cAMP in the pipette, INa averaged 40 pA. In cell-attached patches on tubules from Na-replete rats exposed to cAMP, single Na channels were observed with conductive and kinetic properties similar to those from Na-depleted rats but at lower density. Inclusion of the methyl donor S-adenosyl methionine to the pipette solution did not increase INa in CCTs from Na-replete rats, either in the presence or absence of cAMP. The methylation inhibitor S-adenosyl homocysteine did not affect INa in CCT from Na-depleted animals.

The effect of increased salt intake on blood pressure of chimpanzees.

A colony of 26 chimpanzees given a fruit and vegetable diet of very low Na and high K intake were maintained in long-standing, socially stable small groups for three years. Half of them had salt added progressively to their diet during 20 months. This addition of salt within the human dietetic range caused a highly significant rise in systolic, mean and diastolic blood pressure. The change reversed completely by six months after cessation of salt. The effect of salt differed between chimpanzees, some having a large blood pressure rise and others small or no rise. These results in the species phylogenetically closest to humans bear directly on causation of human hypertension, particularly in relation to migration of preliterate people, with low Na diet, to a Western urban lifestyle with increased salt intake. The hedonic liking for salt and avid ingestion was apt during human prehistory involving hunter-gatherer-scavenger existence in the interior of continents with a scarcity of salt, but is maladaptive in urban technological life with salt cheap and freely available.

Regulation of apical K and Na channels and Na/K pumps in rat cortical collecting tubule by dietary K.

The patch-clamp technique was used to study the properties and the density of conducting K and Na channels in the apical membrane of rat cortical collecting tubule. The predominant K channel observed in cell-attached patches (SK channels) had an outward single-channel conductance (with LiCl in the pipette) of 10 pS. The inward conductance (with KCl in the pipette) was 42 pS. The channel had a high open probability that increased with depolarization. Kinetic analysis indicated the presence of a single open state and two closed states. Increasing K intake by maintaining animals on a high K diet for 12-16 d increased the number of SK channels per patch by threefold (0.7-2.0/patch) over control levels. In addition, conducting Na-selective channels, which were not observed in control animals, were seen at low density (0.5/patch). These channels had properties similar to those observed when the animals were on a low Na diet, except that the mean open probability (0.84) was higher. In other experiments, the whole-cell patch clamp technique was used to measure Na channel activity (as amiloride-sensitive current, INa) and Na pump activity (as ouabain-sensitive current, Ipump). In animals on a high K diet, INa was greater than in controls but much less than in rats on a low Na diet. Ipump was greater after K loading than in controls or Na-depleted animals. These K diet-dependent effects were not accompanied by a significant increase in plasma aldosterone concentrations. To further investigate the relationship between K channel activity and mineralocorticoids, rats were maintained on a low Na diet to increase endogenous aldosterone secretion. Under these conditions, no increase in SK channel density was observed, although there was a large increase in the number of Na channels (to 2.7/patch). Aldosterone was also administered exogenously through osmotic minipumps. As with the low Na diet, there was no change in the density of conducting SK channels, although Na channel activity was induced. These results suggest that SK channels, Na channels and Na/K pumps are regulated during changes in K intake by factors other than aldosterone.