Dietary magnesium, not calcium, regulates renal thiazide receptor.

This study reports for the first time a relationship between dietary Mg and the renal thiazide-sensitive Na-Cl cotransporter (TZR, measured by saturation binding with 3H-metolazone). Ion-selective electrodes measured plasma ionized magnesium (PMg++), calcium (PCa++), and potassium (PK+). Restricting dietary Mg for 1 wk decreased PMg++ 18%, TZR 25%, and renal excretion of magnesium (UMg) and calcium (UCa) more than 50% without changing PCa++, PK+, or plasma aldosterone. A low Mg diet for 1 d significantly decreased PMg++, TZR, UMg and UCa. Return of dietary Mg after 5 d of Mg restriction restored PMg++ and TZR toward normal. In the control, Mg-deficient, and Mg-repleting animals, TZR correlated with PMg++ (r = 0.86) and with UMg (r = 0.87) but not UCa (r = 0.09). Increasing oral intake of Mg for 1 wk increased PMg++ 14%, TZR 32%, UMg 74%, and UCa more than fourfold without changing PCa++ or PK+. In contrast, increasing dietary Ca content from 0.02% to 1.91% did not change TZR, but increased UCa fivefold without changing PCa++. Hormonal mediators (if any) involved in the relationship between dietary Mg and TZR remain to be elucidated, as does the relationship between TZR and tubular reabsorption of Mg.

Genetic control of renal thiazide receptor response to dietary NaCl and hypertension.

Excess NaCl increases blood pressure in some strains of animals but not others. An 8% NaCl diet did not change renal thiazide receptor (TZR) density in two salt-resistant normotensive rat strains (Wistar-Kyoto and Sprague-Dawley) [Fanestil, D. D., D. A. Vaughn, and P. Blakely. Am. J. Physiol. 273 (Regulatory Integrative Comp. Physiol. 42): R1241-R1245, 1997]. However, the renal response to salt differs in normal and hypertensive kidneys [Rettig, R., N. Bandelow, O. Patschan, B. Kuttler, B. Frey, and A. Uber. J. Hum. Hypertens. 10: 641-644, 1996]. Therefore, we examined two strains with salt-aggravated hypertension. Renal TZR did not change when Dahl-S (salt sensitive) animals became hypertensive with 8% dietary NaCl. In contrast, renal TZR decreased 34%, whereas blood pressure increased further, in SHR with 8% dietary NaCl. Blood pressure increased after NG-nitro-L-arginine in SHR, but renal TZR did not change, indicating the salt-induced decrease in TZR in SHR cannot be attributed nonspecifically to elevated arterial pressure. We conclude that the renal response to NaCl-induced increases in blood pressure can be genetically modulated independently of the genes that mediate either the primary hypertension or the salt sensitivity of the hypertension. This finding may be of use in future studies directed at identifying genotypes associated with salt-dependent hypertension.

Dietary NaCl and KCl do not regulate renal density of the thiazide diuretic receptor.

We tested the postulate that the renal density of the thiazide-inhibitable Na-Cl cotransporter or thiazide receptor (TZR) is modulated as part of the renal homeostatic response to changes in dietary intake of NaCl or KCl. Renal excretion of NaCl or KCl varied > 10-fold in response to alterations in oral intake. Renal TZR density was quantitated by binding of [3H]metolazone to renal membranes. Renal TZR density was not altered by sodium deficit (with increased plasma aldosterone concentration), by sodium surfeit (8% NaCl content of diet), by potassium deficit (with hypokalemia), or by potassium surfeit (drinking 1% KCl solution). Unexpectedly, we conclude that regulation of the renal density of TZR is not part of the renal homeostatic responses that adjust excretion of NaCl and KCl to changes in dietary intake of NaCl or KCl.

Metabolic acid-base influences on renal thiazide receptor density.

The renal responses to metabolic acidosis/alkalosis involve changes in the proximal tubule, loop of Henle, and collecting ducts. We tested for acid- or base-induced changes in the distal convoluted tubule (DCT) by examining the renal density of the DCT's receptor for thiazide-type diuretics (TZR), as estimated by the binding of [3H]metolazone in Wistar-Kyoto rats. TZR density significantly decreased by 17% in rats ingesting NH4Cl for 3.5 days and by nearly 30% after 7 days; TZR increased up to 40% in rats ingesting NaHCO3 for 2-4 days but was no longer significantly increased after 7 days. Urinary excretion of chloride increased as renal density of the TZR decreased, a finding consistent with the interpretation that acidosis/alkalosis not only altered TZR density but coordinately altered reabsorption of NaCl by the thiazidesensitive Na-Cl cotransporter. The result is that delivery of Na from DCT is enhanced during acidosis and decreased during alkalosis, assisting in compensatory changes in distal nephron secretion of hydrogen ion. The integrated renal response to metabolic acidosis/alkalosis involves a decrease in renal TZR with acidosis and an increase in TZR with alkalosis.

Thiazide diuretics normalize urinary calcium in spontaneously hypertensive male rats.

The role of the thiazide-sensitive distal convoluted tubule (DCT) in the hypercalciuria of the spontaneously hypertensive rat (SHR) strain was examined by determining (a) the renal density of the thiazide diuretic receptor with 3H-metolazone, and (b) the renal response to a maximal dose of bendroflumethiazide (BFTZ). We confirm that the renal thiazide receptor density was greater in SHR than WKY (0.936 +/- 0.026 vs. 0.797 +/- 0.045 pmol/mg protein; P = 0.02). Prior to BFTZ the urinary excretion of calcium (0.525 +/- 0.061 vs. 0.274 +/- 0.049 micromol per micromol creatinine, P < 0.01) and sodium (12.6 +/- 1.27 vs. 7.89 +/- 0.926 micromol per micromol creatinine; P < 0.01) were greater in SHR versus WKY. BFTZ decreased the excretion of calcium only in SHR and to a level (0.250 +/- 0.032) not significantly different (P = 0.519) from WKY (0.225 +/- 0.032). Surprisingly, BFTZ increased chloride excretion to a greater extent in WKY than in SHR (P = 0.008). We postulate that hypercalciuria in SHR is a manifestation of incomplete uptake of calcium from the tubule lumen across the apical cell membrane in the DCT of the SHR nephron.

Amylin, calcitonin gene-related peptide, and adrenomedullin: effects on thiazide receptor and calcium.

We previously reported that salmon calcitonin, but not rat calcitonin, increased renal thiazide receptor (TZR) density and decreased renal calcium [urinary calcium excretion (U(Caex))] in the rat. Since calcitonins, islet amyloid polypeptide (amylin), calcitonin-gene related peptide (CGRP), and adrenomedullin interact with a family of calcitonin-related receptors, we examined the effects of these peptides on 1) TZR density, as quantitated by binding of [3H]metolazone to renal membranes; 2) plasma ionic composition; and 3) urinary electrolyte excretion. Subcutaneous amylin both increased TZR density nearly twofold and decreased U(Caex), with maximal effects by 24 h. The decreased U(Caex) occurred with plasma amylin levels in the physiological range, whereas the increased TZR did not reach maximum even with plasma amylin >100 times above normal. Similar doses of adrenomedullin increased TZR density modestly but without effect on U(Caex), whereas CGRP did not alter TZR density and tended to increase U(Caex). We propose that U(Caex) and TZR density in the rat kidney are regulated by rat amylin but not by rat calcitonin.

Effects of calcium-modulating hormones on thiazide receptor density.

Thiazide diuretic drugs act in the distal convoluted tubule (DCT) to inhibit a Na+Cl- cotransporter and enhance reabsorption of luminal calcium. The density of receptors for thiazides in the rat DCT is known to be increased by adrenocortical steroids, furosemide, and bendroflumethiazide, but decreased by ischemia. Because the DCT is a physiologic site of action by calcitonin and parathyroid hormone, this study examined the effects of these calcitropic hormones in thyroparathyroidectomized Sprague-Dawley rats on (1) the density of the rat thiazide receptor (TZR), as quantitated by binding of (3H)metolazone to renal membranes, and (2) urinary electrolyte excretion rate. Salmon calcitonin (sCT) (20 to 100 ng/h) (1) increased the density of the renal TZR twofold, an effect that is maximal by 6 h after sCT administration, and (2) decreased urinary calcium excretion rate. Adequate dietary calcium must be provided for the effects of sCT to be observed. Regression analysis demonstrated that renal TZR density correlated negatively with total urinary calcium excretion rate but not with plasma calcium ion concentration. In addition, neither rat calcitonin (rCT), at doses that cause hypocalcemia, nor parathyroid hormone, at doses that cause hypercalcemia, produce direct effects on TZR density in the DCT of the thyroparathyroidectomized rat. Our findings indicate that upregulation of TZR by sCT, which occurs independently of plasma calcium-ion concentration, is likely via a calcitonin-like receptor other than that for rat calcitonin itself.

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.

Influence of gender on renal thiazide diuretic receptor density and response.

The influence of gender and gonadectomy on (1) the density of the renal thiazide-sensitive ion transporter, as quantitated by the ability of renal membranes to bind (3H)metolazone, and (2) the changes in the urinary excretion of electrolytes caused by maximal bendroflumethiazide (BFTZ) in Sprague-Dawley rats was determined. The density of the thiazide receptor was twofold higher (P < 0.001) in females than in males. Orchiectomy increased thiazide receptor significantly in one of two studies (P < 0.01). Ovariectomy decreased thiazide receptor by more than 20% (P < 0.01) in both studies. The rates of the urinary excretion of sodium and chloride after BFTZ and the increases in the urinary excretion of sodium, chloride, and ammonium caused by BFTZ were greater in intact females than in intact males; BFTZ decreased the urinary excretion of calcium 50% in intact females, but not in intact males. Regression analysis of the thiazide receptor (in intact and gonadectomized animals) versus the urinary excretion of electrolytes before and after BFTZ yielded a model in which one-third of the variation in thiazide receptor could be related to the change in the excretion of calcium and ammonium produced by BFTZ, raising the possibility that the density of the thiazide receptor might be related to calcium or acid-base homeostasis. It was concluded that the renal excretion of sodium, chloride, calcium, and ammonium are, in part, controlled by gender and sex hormones via their regulation of the renal density of the thiazide diuretic receptor.

Inhibition of binding of [3H]metolazone to rat kidney membrane by stilbene disulfonates.

Thiazide diuretics inhibit an electroneutral chloride and sodium reabsorptive transport pathway in the renal distal convoluted tubule. [3H]Metolazone binds with high affinity to the drug recognition site on the thiazide-sensitive ion transporter. The molecular nature of this transporter is currently unknown. This report examines whether stilbene disulfonates [4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS)], agents that have been used to identify other anion-transporting proteins, could be useful in the identification of the thiazide receptor. We found that high concentrations of DIDS or SITS were required to inhibit binding of [3H]metolazone to rat kidney membranes, with apparent IC50 values of 380 and 940 microM, respectively. The inhibition was due to a decrease in the number of binding sites without alteration in the affinity of the binding. The inhibition was not reversible, as judged by the inability of the inhibition to be reversed by removal of the DIDS or SITS from the membranes prior to equilibration with [3H]metolazone. Addition of 100 mM NaCl to the incubation medium did not protect the [3H]metolazone binding site from inhibition by DIDS. We infer that DIDS and SITS irreversibly inhibit binding of [3H]metolazone by reaction with the thiazide receptor at a site other than the anion-transporting site.

Effects of diuretic treatment and of dietary sodium on renal binding of 3H-metolazone.

We report a series of experiments designed to determine if agents and conditions that have been reported to alter sodium reabsorption, Na-K-ATPase activity or cellular structure in the rat distal nephron might also regulate the density or affinity of binding of 3H-metolazone to the putative thiazide receptor in the distal nephron. Experimental conditions selected for study were acute (60-min) and chronic hydrochlorothiazide (HCTZ), acute acetazolamide, acute and chronic furosemide, and 14 days of varied intake of dietary sodium. The density of the binding of 3H-metolazone was increased 47% by acute HCTZ (P less than 0.001) and 39% (P less than 0.001) by acute furosemide. In contrast, acute acetazolamide produced no change in binding despite eliciting a dramatic diuresis. Chronic HCTZ (5 days) and chronic furosemide (7 days) increased binding of 3H-metolazone by 46% (P less than 0.001) and by 101% (P less than 0.001), respectively. Variation of dietary sodium intake over a range that allowed normal growth of the animal and that produced urinary excretion of Na varying from 0.28 to 2.62 mEq/100 g/day failed to alter the density of binding of 3H-metolazone. These studies are the first indication that the density of the thiazide receptor is regulated by a variety of both acute and chronic conditions that have previously been associated with changes in transport, ultrastructure or Na-K-ATPase activity in the distal nephron.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of ions on binding of the thiazide-type diuretic metolazone to kidney membrane.

The effect of a number of ions on the binding of the thiazide-type diuretic metolazone (MTZ) to rat renal cortical membranes was studied to elucidate the mechanism of NaCl transport in the kidney distal tubule. Among the cations tested, Na+ significantly stimulated the binding up to 2.4-fold over control. The effective concentration of Na+ that produced half-maximal stimulation was 2-17 mM. Li+, K+, NH4+, Rb+, and Cs+ produced little stimulation of binding of MTZ. Several anions including Cl- inhibited binding. The inhibition of binding of MTZ by Cl- was enhanced by Na+ and Li+. Scatchard analyses revealed that 50 mM Na+ increased the affinity for binding of MTZ from a Kd = 3.56 +/- 0.15 nM to Kd = 1.32 +/- 0.11 nM. Chloride, in the presence of 50 mM Na+, competitively inhibited binding of MTZ by suppressing the affinity to Kd = 9.27 +/- 1.11 nM without changing the maximal number of binding sites (0.733 +/- 0.049 pmol/mg). A mechanism for the MTZ-sensitive NaCl transport is proposed, in which the transporter protein possesses a binding site for Na+ and a binding site for Cl-, which is also the binding site for MTZ. Na+ binds to its site and increases the affinity for Cl-/MTZ. The binding of Cl- to the transporter enables the import of Na+ and Cl- across the tubule membrane. MTZ, however, when present competes with Cl- for the binding site on the transporter and prevents the transport of Na+ and Cl-.

Solubilization of thiazide diuretic receptors from rat kidney membranes.

Thiazide-type diuretics act at receptors to inhibit NaCl transport in the renal distal tubule. We solubilized high-affinity [3H]metolazone binding sites from rat kidney membranes with Triton X-100, which was more effective than several other detergents. Phosphatidylcholine and a mixture of proteinase inhibitors were needed to stabilize binding so that 57% of solubilized binding remained after 72 h at 4 degrees C. The affinities of solubilized (Kd = 11.4 +/- 0.5 nM) and membrane-bound receptors (Kd = 12.0 +/- 1.7 nM) were similar. The maximal number of binding sites/mg protein of solubilized receptors was 46 +/- 3% (n = 5) of membrane-bound receptors. Diuretics with a wide range of affinities had similar affinities for the solubilized and membrane-bound sites. Chloride inhibited and sodium stimulated the binding of [3H]metolazone to solubilized receptors, as they do with membrane-bound receptors. These studies demonstrate that, as judged by ligand binding, thiazide receptors can be solubilized in an active conformation and provide the basis for future purification and reconstitution.