Salt-sparing diuretic action of a water-soluble urea analog inhibitor of urea transporters UT-A and UT-B in rats.

Inhibitors of kidney urea transporter (UT) proteins have potential use as salt-sparing diuretics ('urearetics') with a different mechanism of action than diuretics that target salt transporters. To study UT inhibition in rats, we screened about 10,000 drugs, natural products and urea analogs for inhibition of rat UT-A1. Drug and natural product screening found nicotine, sanguinarine and an indolcarbonylchromenone with IC50 of 10-20 µM. Urea analog screening found methylacetamide and dimethylthiourea (DMTU). DMTU fully and reversibly inhibited rat UT-A1 and UT-B by a noncompetitive mechanism with IC50 of 2-3 mM. Homology modeling and docking computations suggested DMTU binding sites on rat UT-A1. Following a single intraperitoneal injection of 500 mg/kg DMTU, peak plasma concentration was 9 mM with t1/2 of about 10 h, and a urine concentration of 20-40 mM. Rats chronically treated with DMTU had a sustained, reversible reduction in urine osmolality from 1800 to 600 mOsm, a 3-fold increase in urine output, and mild hypokalemia. DMTU did not impair urinary concentrating function in rats on a low protein diet. Compared to furosemide-treated rats, the DMTU-treated rats had greater diuresis and reduced urinary salt loss. In a model of syndrome of inappropriate antidiuretic hormone secretion, DMTU treatment prevented hyponatremia and water retention produced by water-loading in dDAVP-treated rats. Thus, our results establish a rat model of UT inhibition and demonstrate the diuretic efficacy of UT inhibition.

Electrical conductivity measurements of urine as a new simplified method to evaluate the diuretic activity of medicinal plants.

ETHNOPHARMACOLOGICAL RELEVANCE: Diuretic plants are widely used in traditional medicine in many countries. However, many of these species have not been subjected to experimental studies to confirm that property. In this paper, a simple new method is proposed to evaluate the diuretic activity of plants. We define a new index that takes into account only the volume of urinary excretion and total ion concentration excreted obtained by specific electrical conductivity measurements. MATERIALS AND METHODS: Urine was collected in a graduate cylinder during the 8h after Artemisia thuscula (AT), Withania aristata (WA), Smilax canariensis (SC) and HCTZ oral administration to laboratory mice. To obtain the new index Diuretic Power (DP), we measured the specific electrical conductivity (κ) of the fresh urine samples. We calculated the concentration of a NaCl (or KCl) aqueous solution that has the same specific electrical conductivity as the urine sample. We multiplied this concentration by the corresponding urinary excretion volume, thus obtaining the total mEq. of electrolyte excreted "as if all were NaCl (or KCl)". Finally, we divided these mEq. by those corresponding to the control to obtain the DP value. RESULTS: HCTZ showed a 40% increase in DP, with respect to the control group, independently of the doses used, and the studied plants produced an increase between 7 and 28%. DP values were compared with other common indexes, DI and SIi, showing that the variation sequence of the three indexes was the same for HCTZ, WA and SC. CONCLUSIONS: A new and easy index, that we called diuretic power (DP), for estimating the diuretic activity of drugs or plants is proposed. It allows us to highlight diuretic effect with respect to a control value of a large amount of drugs or plants that had not been previously experimentally studied.

The effect of saponins from Ampelozizyphus amazonicus Ducke on the renal Na+ pumps' activities and urinary excretion of natriuretic peptides.

BACKGROUND: In a previous study, we showed that a saponin mixture isolated from the roots of Ampelozizyphus amazonicus Ducke (SAPAaD) reduces urine excretion in rats that were given an oral loading of 0.9 % NaCl (4 ml/100 g body weight). In the present study, we investigated whether atrial natriuretic peptides (ANP) and renal ATPases play a role in the SAPAaD- induced antidiuresis in rats. METHODS: To evaluate the effect of SAPAaD on furosemide-induced diuresis, Wistar rats (250-300 g) were given an oral loading of physiological solution (0.9 % NaCl, 4 ml/100 g body weight) to impose a uniform water and salt state. The solution containing furosemide (Furo, 13 mg/kg) was given 30 min after rats were orally treated with 50 mg/kg SAPAaD (SAPAaD + Furo) or 0.5 ml of 0.9 % NaCl (NaCl + Furo). In the SAPAaD + NaCl group, rats were pretreated with SAPAaD and 30 min later they received the oral loading of physiological solution. Animals were individually housed in metabolic cages, and urine volume was measured every 30 min throughout the experiment (3 h). To investigate the role of ANP and renal Na(+) pumps on antidiuretic effects promoted by SAPAaD, rats were given the physiological solution (as above) containing SAPAaD (50 mg/kg). After 90 min, samples of urine and blood from the last 30 min were collected. Kidneys and atria were also removed after previous anesthesia. ANP was measured by radioimmunoassay (RIA) and renal cortical activities of Na(+)- and (Na(+),K(+))-ATPases were calculated from the difference between the [32P] Pi released in the absence and presence of 1 mM furosemide/2 mM ouabain and in the absence and presence of 1 mM ouabain, respectively. RESULTS: It was observed that SAPAaD inhibited furosemide-induced diuresis (at 90 min: from 10.0 ± 1.0 mL, NaCl + Furo group, n = 5, to 5.9 ± 1.0 mL, SAPAaD + Furo group n = 5, p < 0.05), increased both Na(+)-ATPase (from 25.0 ± 5.9 nmol Pi.mg(-1).min(-1), control, to 52.7 ± 8.9 nmol Pi.mg(-1).min(-1), p < 0.05) and (Na(+),K(+))-ATPase (from 47.8 ± 13.3 nmol Pi.mg(-1).min(-1), control, to 79.8 ± 6.9 nmol Pi .mg(-1).min(-1), p < 0.05) activities in the renal cortex. SAPAaD also lowered urine ANP (from 792 ± 132 pg/mL, control, to 299 ± 88 pg/mL, p < 0.01) and had no effect on plasma or atrial ANP. CONCLUSION: We concluded that the SAPAaD antidiuretic effect may be due to an increase in the renal activities of Na(+)- and (Na(+),K(+))-ATPases and/or a decrease in the renal ANP.

Diuretic activity of some Smilax canariensis fractions.

ETHNOPHARMACOLOGICAL RELEVANCE: Smilax canariensis is an endemic species of the Canary Islands, popularly known as "Zarzaparrilla sin espinas". The rhizome, leaves and stem of this species has wide use in folk medicine practice on the islands, where they are habitually employed as diuretic. AIM OF THE STUDY: In this paper we report on the diuretic activity in experimental animals of several fractions of the methanol extract of this species. MATERIALS AND METHODS: Four fractions of the methanol extract of the rhizomes, leaves and stem of Smilax canariensis (50 and 100mg/kg), Furosemide and Hydrochlorotiazide (10mg/kg), were orally administered to laboratory animals to evaluate their diuretic activity. Water excretion rate, pH, conductivity, and content of Na(+), K(+) and Cl(-) were measured in the urine of saline-loaded mice. RESULTS: All the studied extracts showed an interesting increase of the diuresis, although the n-buthanol (27%; p<0.05) and ethyl acetate extract (35%; p<0.01), at 100mg/kg p.o., showed the most interesting diuretic activity, which suggested that this diuretic effect is associated with the compounds contained in the fractions of intermediate polarity (ethyl acetate and n-butanol), decreasing in the most extreme apolar and polar sub-extracts (dichloromethane and methanol: water respectively). The increase in diuresis produced by these two extracts was very close to the values of Hydrochlorothiazide (32%) or Furosemide (39%), used as reference diuretics. CONCLUSIONS: These data, together with previous results on the aqueous and methanol extracts, reaffirm assertions made regarding the effectiveness of the extracts of this plant against urinary pathologies in the Canary Islands folk medicine.

[Pseudohypoaldosteronism in infants with salt wasting syndrome. Two case reports]. / Pseudohipoaldosteronizm u niemowlat jako przyczyna zespolu utraty soli. Opis dwóch przypadków.

The paper discusses two cases of male infants, who developed a markedly elevated salt wasting syndrome in early infancy, resistant to treatment with mineralocorticoids. Steroid urinary profiles excluded congenital adrenal hyperplasia. However, both patients presented with extremely high excretion of aldosterone metabolite THAldo without effects of aldosterone action, what resulted in pseudohypoaldosteronism (PHA) diagnosis. The patients were treated with sodium supplementation, which normalized their clinical state and serum electrolytes. In the first patient the sporadic form of renal PHA1 is suspected. In the second patient congenital urinary tract anomalies and infection resulted in transient PHA1. Pseudohypoaldosteronism should be considered in the differential diagnosis of a salt wasting syndrome in infants, especially when it is accompanied by infections or congenital defects of the urinary tract.

Urinary level of 1,N(6) -ethenodeoxyadenosine, a marker of oxidative stress, is associated with salt excretion and omega 6-polyunsaturated fatty acid intake in postmenopausal Japanese women.

Excretion of 1,N(6)-ethenodeoxyadenosine (epsilon dA), a marker for lipid peroxidation (LPO)-derived DNA damage was analyzed in urine of nonsmoking postmenopausal women participating in a dietary intervention trial in Northern Japan. Hereby the efficacy of dietary consultation in reducing salt and increasing vitamin C and carotenes during 1 year was estimated. Thirty postmenopausal women, 60-69 years of age, from the intervention group and 30 age-matched women from the control group were randomly selected. The subjects completed a self-administered diet history questionnaire and in the pre- and post-intervention period 48 hr urine and fasting blood samples were collected. epsilon dA in urine was analyzed by an immuno-precipitation-high performance liquid chromatography-fluorescence detection method. epsilon dA excretion (/48 hr) in the 59 postmenopausal Japanese women with complete urine collection ranged from 12-226 pmol at the pre-intervention. At the pre-intervention, epsilon dA excretion was positively associated with urinary salt excretion (R = 0.33, p = 0.01) and omega-6 polyunsaturated fatty acid intake (%energy value, R = 0.28, p = 0.03) in the 59 women. The average epsilon dA excretion in the intervention group was 61 pmol at pre-intervention and 44 pmol at post-intervention (p = 0.14). In the control group, it was 58 pmol at pre-intervention and 75 pmol at post-intervention (p = 0.24). During the intervention period, 18/29 (62%) of the subjects in the intervention group exhibited the decreased excretion and 10/26 (38%) in the control group (p = 0.08). Results from this pilot study suggest urinary epsilon dA as a potential biomarker of DNA damage possibly derived from salt-induced inflammation and LPO; further exploration of epsilon dA in human biomonitoring studies is warranted.

Renal effects of serine-7 analog of lymphoguanylin in ex vivo rat kidney.

Guanylin and uroguanylin compose a family of natriuretic, diuretic, and kaliuretic peptides that bind to and activate apical membrane receptor guanylyl cyclase signaling molecules in renal and intestinal epithelia. Recently, a complementary DNA encoding an additional member of the guanylin family of cGMP-regulating peptides was isolated from lymphoid tissues of the opossum and was termed lymphoguanylin (LGN). A peptide analog of opossum LGN was synthesized containing a single disulfide bond with the internal cysteine-7 replaced by a serine residue (LGN(Cys7-->Ser7)). The biological activity of LGN(Ser) was tested by using a cGMP bioassay with cultured T84 (human intestinal) cells and opossum kidney (OK) cells. LGN(Ser) has potencies and efficacies for activation of cGMP production in the intestinal and kidney cell lines that are 100- and 1,000-fold higher than LGN, respectively. In the isolated perfused rat kidney, LGN(Ser) stimulated a maximal increase in fractional Na+ excretion from 24.8 +/- 3.0 to 36.3 +/- 3.3% 60 min after administration and enhanced urine flow from 0.15 +/- 0.01 to 0.24 +/- 0.01 ml. g(-1). min(-1). LGN(Ser) (0.69 microM) also increased fractional K+ excretion from 27.3 +/- 2.3 to 38.0 +/- 3.0% and fractional Cl- excretion from 26.1 +/- 0.8 to 43.5 +/- 1.9. A ninefold increase in the urinary excretion of cGMP from 1.00 +/- 0.04 to 9.28 +/- 1.14 pmol/ml was elicited by LGN(Ser), whereas cAMP levels were not changed on peptide administration. These findings demonstrate that LGN(Ser), which contains a single disulfide bond like native LGN, activates guanylyl cyclase-C (GC-C) receptors in T84 and OK cells and may be very helpful in studying the physiological importance of activation of GC-C in vivo. LGN(Ser) also exhibits full activity in the isolated perfused kidney equivalent to that observed previously with opossum uroguanylin, suggesting a physiological role for LGN in renal function. Thus the single amino acid substitution enhances the activity and potency of LGN.

Adrenocortical steroids increase renal thiazide diuretic receptor density and response.

The density of the rat renal pharmacologic receptor for thiazide-type diuretics, as quantitated by the maximal specific binding of (3H)metolazone, decreased to one-third normal after adrenalectomy. Selective glucocorticoid (dexamethasone or RU-28362) replacement increased thiazide receptor density to or above the normal level over the dose range of steroid that decreased thymus weight, which served as a bioassay for glucocorticoid activity. Mineralocorticoid (fludrocortisone or aldosterone), in doses that did not decrease thymus weight, also increased thiazide diuretic receptor density to or above normal. The addition of glucocorticoid (RU-28362) to maximal aldosterone increased thiazide receptor above that produced by aldosterone alone and to threefold normal. Similarly, the addition of aldosterone to high-dose RU-28362 also increased thiazide receptor density above that produced by the glucocorticoid alone and to threefold normal. Hence, the effects of glucocorticoids and mineralocorticoids appeared to be additive. The increase in renal thiazide receptor density produced by fludrocortisone, at a dose that elicited both mineralocorticoid and glucocorticoid effects, was unrelated to the basal (prethiazide) renal excretion of sodium, potassium, chloride, or calcium. However, fludrocortisone-pretreated animals responded to bendroflumethiazide with a greater natriuresis than did controls. In addition, the magnitudes of the thiazide-elicited natriuresis and chloriuresis correlated significantly with thiazide receptor. It was concluded that both the density of the renal thiazide receptor and the quantity of sodium and chloride reabsorbed by the thiazide-sensitive Na-Cl cotransporter in the kidney are under adrenocortical regulation.

Sodium chloride supplement at diagnosis and during infancy in children with salt-losing 21-hydroxylase deficiency.

Eight infants (6 female, 2 male) with salt-losing congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency were studied to determine the sodium deficit at diagnosis and the level of salt supplement required in addition to subsequent hormone replacement. The median sodium deficit at diagnosis was 34 mmol (range 16-78) or 10.5 mmol/kg (range 4-24). A mean sodium supplement of 2.2 mmol/kg per day (range 0.5-4.9), double the amount provided with feeds, was required to maintain plasma sodium concentration and plasma renin activity (PRA) in the normal range for age. We present an equation based on sodium output (urine), sodium input (feeding plus supplement) and plasma sodium concentration to calculate the sodium supplement needed to maintain sodium balance on hormone replacement in this condition and some practical management suggestions. The necessity for salt supplements is often underestimated and the salt-losing tendency exacerbated by infection remains an unnecessary reason for hospitalization during the first months of life. In patients with salt-losing CAH life-long mineralocorticoid treatment is necessary but additional salt supplements are needed to maintain plasma sodium concentration and PRA in the normal range during infancy.

Pseudohypoaldosteronism.

10 infants are described with pseudohypoaldosteronism, 5 in detail and a further 5 briefly. They all presented with hyperkalaemia, urinary salt-wasting disease, and ostensibly normal renal and adrenocortical function. Diagnosis was established by demonstrating the greatly increased values of plasma renin activity and plasma aldosterone concentration, plus the increased excretion of aldosterone and its metabolites on gas chromatographic and mass spectrometric analyses of urine. The children were treated with sodium chloride supplements, up to 60 mmol/day, but by the time most of the infants were about a year old these could be stopped. Exogenous mineralocorticoids were without effect in those to whom they were administered. The precise aetiology of the condition remains conjectural; lack of renal tubular response to aldosterone seems probable. Pseudohypoaldosteronism may be more common than has been thought and new techniques for investigating salt-wasting disorders may show its true incidence.

Prevention of human deconditioning during prolonged immersion in water.

A 56-day immersion experiment in which two subjects participated was carried out. During the experiment the preventive effect of periodic acceleration combined with exercise and water-salt intake was assessed. Simulating an increased gravitational field, exposure to acceleration increased the static component of the load upon the musculo-skeletal system, increased the gradient of the blood hydrostatic pressure, activated mechanisms responsible for the venous return to the heart, stimulated systems regulating antidiuretic and antisodiumdiuretic reflexes. Involvement of these mechanisms restored haemodynamic parameters, fluid-electrolyte balance and blood coagulability. The prophylactic effect of acceleration was enhanced if combined with exercise and supplemented water-salt intake.