Thiazide Receptor Research Articles

Abstract P209: Antihypertensive Medication Non-Adherence is Common in Patients With Suspected Refractory Hypertension

Introduction: Refractory hypertension is a phenotype of antihypertensive treatment failure defined as uncontrolled BP (> 135/85 mmHg) despite the use of five or more different antihypertensive agents, including long-acting thiazide like diuretic (chlorthalidone) and mineralocorticoid receptor antagonist (Spironolactone or Eplerenone). Recently white coat effect has been shown to be uncommon in patients with refractory hypertension. However, the degree of medication non-adherence, an important cause of apparent refractory hypertension, is unknown. Methods: In this prospective evaluation, 37 refractory hypertensive patients were recruited from the University of Alabama at Birmingham Hypertension Clinic after having uncontrolled BP at three or more clinic visits. All patients were evaluated by automated office BP (AOBP) with the BpTRU device, ambulatory BP monitoring (ABPM), and 24-hr urine collection to detect antihypertensive medication adherence by high-performance liquid chromatography-tandem mass spectrometry. Out of 34 patients who underwent ABPM monitoring, 30 (88%) patients were refractory hypertensive by AOBP and by ABPM. Of these, 27 patients had 24-hr urine collection for detection of prescribed antihypertensive medications or their metabolites. Results: Out of 27 patients, 11 (40.7%) patients had complete adherence with five or more antihypertensive medications, indicating true refractory hypertension. Ten (37.0%) patients were partially adherent, and 6 (22.2%) patients had total non-adherence to their antihypertensive medications. Conclusion: Of patients identified as having apparent refractory hypertension, 40% were adherent with at least 5 of their prescribed antihypertensive agents, confirming true refractory hypertension. The remaining 60% were partially or fully non-adherent. These results indicate that non-adherence is common in patients suspected of having refractory hypertension.

Low dose of hydrochlorothiazide, in combination with angiotensin receptor blocker, reduces blood pressure effectively without adverse effect on glucose and lipid profiles

Previous studies have shown highly effective lowering of blood pressure with thiazide diuretics in combination with angiotensin receptor blockers. However, thiazide diuretics may cause the development of diabetes and abnormal lipid metabolism. Little is known as to whether dysmetabolic potential of thiazide diuretics could be neutralized when adding angiotensin receptor blockers. This study consisted of 26 patients with essential hypertension. Patients were randomized to 24 weeks of treatment with either candesartan, 12 mg monotherapy (n = 13, group A), or hydrochlorothiazide (HCTZ), 6.25 mg in combination with candesartan, 8 mg (n = 13, group B). Before and after treatment, we assessed glucose and lipid profiles including adiponectin, resistin, and active glucagon-like peptide-1 (GLP-1) levels. At baseline, there were no differences in age, body mass index, systolic blood pressure (SBP), and diastolic blood pressure (DBP), as well as plasma levels of hemoglobin A1c, insulin, low-density lipoprotein cholesterol, triglycerides, adiponectin, resistin, and active GLP-1 between the two groups. There were significant reductions in SBP (from 152 ± 10 mmHg at baseline to 134 ± 12 mmHg after treatment) and DBP (from 84 ± 5 mmHg at baseline to 71 ± 8 mmHg after treatment) in group A. There were also significant reductions in SBP (from 148 ± 10 at baseline to 128 ± 7 mmHg after treatment) and DBP (from 90 ± 9 at baseline to 74 ± 12 mmHg after treatment) in group B. There were no differences in reduction of SBP or DBP after 24 weeks of treatment between the two groups. There were no changes of the glucose and lipid profiles, including adiponectin, resistin, insulin, and active GLP-1 levels after 24 weeks of treatment in both groups. A low dose of HCTZ in combination with candesartan reduces blood pressure effectively without adverse effects on the glucose and lipid profiles. Therefore, the combination of thiazide diuretics and angiotensin receptor blockers could assist patients in achieving long-term control of blood pressure with good tolerability.

The Impact of Primary Care Dual-Management on Quality of Care

Discontinuity is common in US healthcare. Patients access multiple systems of care and in the nation's largest integrated healthcare system, Veteran's Administration (VA) patients frequently use non-VA primary care providers. The impact of this "dual-management" on quality is unknown. The authors' objective was to identify dual-management and associations with markers of care quality for hypertension and associated conditions. Data was collected via surveys and chart reviews of primary care patients with hypertension from six VA clinics in Iowa and Minnesota. Clinical measures abstracted included the following: goal blood pressure (BP) and use of guideline-concordant therapy, low-density lipoprotein (LDL) cholesterol, hemoglobin A1C, and body mass index (BMI). Dual-management data was obtained through self-report. Of 189 subjects (mean age = 66), 36% were dual-managed by non-VA providers. There was no difference in hypertension quality of care measures by dual-management status. A total of 51% were at BP goal and 58% were on guideline-concordant therapy. Dual-managed patients were more likely to use thiazide diuretics (43% vs 29%; P = .03) and angiotensin receptor blockers (13% vs 3%; P < .01), but less likely to use angiotensin-converting enzyme inhibitors (43% vs 61%; P = .02). There was no difference in LDL cholesterol (97.1 mg/dl vs 100.1 mg/dl; P = .55), hemoglobin A1C (7% vs 6%; P = .74), or BMI (29.8 vs 30.9; P = .40) for dual-managed versus VA managed patients, respectively. Although dual-management may decrease continuity, VA/private sector dual-management did not impact quality of care, though some medication differences were observed. With the high prevalence of dual-management, future work should further address quality and evaluate redundancy of services.

Lack of effect of cicletanine and its sulpho-conjugated metabolite on the thiazide receptor expressed in Xenopus oocytes

1Universidad Nacional de Mexico, Mexico, and INSERM U400, Faculté de Médecine de Créteil and IPSEN, Paris, France

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.

Immunocytochemical characterization of the high-affinity thiazide diuretic receptor in rabbit renal cortex.

Thiazide diuretics increase urinary NaCl excretion primarily by inhibiting Na and Cl transport across the apical membrane of cells in the renal distal tubule. Although these diuretics bind to a membrane protein that couples transport of Na and Cl directly, the molecular nature of this transporter and its localization in the mammalian kidney remain controversial. The present experiments were designed to develop monoclonal antibodies to the high-affinity thiazide diuretic receptor to investigate its molecular characteristics and its cellular and subcellular localization in rabbit kidney. Mice were immunized with high-affinity thiazide diuretic receptors that had been partially purified from rabbit kidney cortex. Resulting hybridomas were screened for the ability to immunoprecipitate thiazide diuretic receptors that were labeled with the thiazide-like diuretic [3H]metolazone. A single hybridoma (MAb JM5) produced antibodies capable of immunoprecipitating up to 80% of the labeled thiazide receptors from solubilized renal cortical membranes. MAb JM5 reacted with a 125-kDa protein on Western blots of solubilized renal cortical apical membranes. It stained the apical membrane of cells in the distal convoluted and connecting tubule but did not stain proximal tubules, glomeruli, or interstitial structures. Less intense staining of apical membranes of principal cells in the collecting tubule and a subpopulation of cells in the thick ascending limb were also present. These results indicate that the high-affinity thiazide diuretic receptor comprises a 125-kDa protein that localizes to the apical membrane of cells in the renal distal tubule.

Steroid regulation of thiazide-sensitive transport.

HE STUDY of regulation by steroid hormones of the reabsorption of sodium and chloride inhibitable by diuretics of the thiazide ( 1,2,4-benzothiadiazine- 1,l -dioxide) class has lagged a decade or more behind studies of steroid action in other portions of the nephron. This tardiness can be attributed primarily to three factors. First, the complexity and structural segmentation of the “distal tubule” had not been appreciated. Second, the segment of the nephron in which thiazide-type diuretics exert their action-pharmacologically identified as “the cortical diluting segment”-appeared to be portion of the nephron that was poorly accessable to micropuncture-the early cortical distal nephron. Third, no radioactive “marker” of the thiazide-sensitive transporter (that is, of the “thiazide receptor”) was available. I shall discuss a current view of each of these areas.

Solubilization and partial purification of the thiazide diuretic receptor from rabbit renal cortex

This study was designed to solubilize, characterize and begin to purify the thiazide-sensitive Na/Cl transporter from mammalian kidney. Metolazone, a thiazide-like diuretic drug, binds to receptors in rat renal cortex closely related to the thiazide-sensitivee Na/Cl transport pathway of the renal distal tubule. In the current study, [ 3H]metolazone bound to receptors in rabbit renal cortical microsomes. The portion of [ 3H]metolazone binding that was inhibited by hydrochlorothiazide reflected binding to a high-affinity class of receptor. The affinity ( K d2.0 ± 0.1nM) and number ( B max = 0.9 ± 0.4pmol/mg protein) of high-affinity receptors in rabbit renal cortical membranes were similar to values reported previously for rat. When proximal and distal tubule fragments were separated by Percoll gradient centrifugation, receptors were restricted to the fraction that contained distal tubules. When compaed with cortical homogenates, receptor density was enriched 12-fold by magnesium precipitation and differential centrifugation. The zwitterionic detergent CHAPS solubilized 25–35% of the receptors (at 6 mM). Chloride inhibited and Na stimulated binding of [ 3H]metolazone to solubilized high-affinity receptors. The receptors could be purified significantly by hydroxyapatite chromatography and size exclusion high performance liquid chromatography (HPLC). The combination of magnesium precipitation and differential centrifugation, hydroxyapatite chromatography, and size exclusion HPLC resulted in a 213-fold enrichment of receptors, compared to renal cortical homogenate. The current indicate that thiazide receptors from rabbit kidney share characteristics with receptors from rat, and that rabbit receptors can be solubilized in CHAPS and purified significantly by hydroxyapatite chromatography and size exclusion HPLC.

Adaptation of distal convoluted tubule of rats. II. Effects of chronic thiazide infusion

Mammalian distal tubules adapt structurally and functionally when NaCl concentration in tubule fluid is altered chronically. These experiments were designed to test the hypothesis that chronic administration of hydrochlorothiazide (HCTZ), a drug that blocks Na and Cl uptake across apical membranes of rat distal tubule cells, would reduce intrinsic transport capacity of distal tubules and reduce the number of thiazide-sensitive transporters. Osmotic pumps were implanted into rats to deliver 3.75 mg/day HCTZ or vehicle for 10-14 days. All animals were offered a solution containing 0.8% NaCl and 0.1% KCl as drinking fluid. Free-flow micropuncture after 10-14 days indicated that Na and Cl delivery to distal tubule was not significantly different in HCTZ- and vehicle-treated animals. Microperfusion in vivo with an artificial interstitial solution, with no thiazide, indicated that 10-14 days of HCTZ infusion did reduce Na transport capacity of distal tubules from 390 +/- 32 to 203 +/- 24 pmol/min (P less than 0.01). In contrast, the number of thiazide-sensitive NaCl transporters, determined as high-affinity receptors for [3H]metolazone in renal cortical membranes, was higher in HCTZ group than in controls (2.2 +/- 0.4 vs. 1.0 +/- 0.1 pmol/mg protein, P less than 0.01). These data support the hypothesis that chronic blockade of NaCl entry across apical membranes of distal tubule cells reduces NaCl transport capacity, an effect that occurs despite an increase in the number of thiazide receptors. They indicate that thiazide receptor binding studies should be interpreted in combination with direct functional measurements.

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 m 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 IC 50 values of 380 and 940 μM, 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)

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°C. The affinities of solubilized ( K d = 11.4 ± 0.5 nM) and membrane-bound receptors ( K d = 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.

Thiazide Diuretic Receptors in Spontaneously Hypertensive Rats and 2-Kidney 1-Clip Hypertensive Rats

Thiazide diuretic receptor density was assessed in kidneys from spontaneously hypertensive rats (SHRs) and normotensive Wistar-Kyoto (WKY) rats by measuring hydroflumethiazide-displaceable 3H-metolazone binding to renal membranes in vitro. Renal thiazide receptor density was not significantly different in 4 week old SHR and WKY rats, but was significantly increased by 20%-40% in 14-49 week old SHRs compared to WKY rats. Affinity of receptors for 3H-metolazone did not differ between SHRs and WKY rats at any age. In WKY rats with 2 kidney-1 clip (2K-1C) hypertension, thiazide receptor density was not significantly different in either clipped or unclipped kidneys from sham-operated controls. Thus, increased renal thiazide receptor density occurs in SHRs along with the development of hypertension and does not appear to be secondary to increased renal perfusion pressure. This increase may reflect altered hormonal or ionic input to the distal tubule and may contribute to elevated sodium reabsorption in this segment in the SHR.