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At least 19 recordsLinked to original sources
PubMed1996

Identification of a cDNA encoding a thiazide-sensitive sodium-chloride cotransporter from the human and its mRNA expression in various tissues.

We report here the identification of a cDNA encoding a human thiazide-sensitive sodium-chloride cotransporter (hTSC) using a PCR-based method. The homology of the hTSC with rat TSC (rTSC) and rat bumetanide-sensitive sodium-potassium-chloride cotransporter (rBSC) was 86% and 64%, respectively, at the nucleotide level, and 92% and 61%, respectively, at the amino acid level. Using fluorescence in situ hybridization (FISH), the hTSC gene has been mapped to chromosome 16q13. Northern blot analysis using polyA+RNA from various human tissues, revealed a major 4.5 kb transcript and a minor 6.5 kb specifically in the kidney, but low level of expression was also observed in small intestine, placenta, prostate, colon, and spleen.

Amino Acid Sequence↗
PubMed1996

Linkage of Gitelman syndrome to the thiazide-sensitive sodium-chloride cotransporter gene with identification of mutations in Dutch families.

Gitelman syndrome is a mostly autosomal recessive disorder affecting the renal tubular function associated with hypokalemia and hypomagnesemia. Functional studies point to a defect in the distal renal tubule in the thiazide-sensitive, electroneutral sodium-chloride co-transporter (TSC). Based upon the localization of a 2.6 cDNA encoding the human TSC to chromosome 16q13, polymorphic markers spanning the region from 16p12 to 16q21 were tested for linkage to the Gitelman syndrome locus in three Dutch families with autosomal recessive inheritance of this disorder. Using two-point linkage analysis, a maximum LOD score (Zmax of 4.49 (at theta = 0.00) was found for the marker D16S408. One crucial recombination event places the Gitelman syndrome locus distal to D16S419 at 16q12-13. Subsequently we have tested our group of Gitelman patients for mutations in the human TSC gene. Two mutations were identified in three Gitelman families. Our study confirms that the human TSC gene is involved in Gitelman syndrome. Patients from three Gitelman families reveal two identical human TSC mutations, suggesting these families share a common ancestor.

Benzothiadiazines↗
PubMed1991

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-sensitive 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 (Kd 2.0 +/- 0.1 nM) and number (Bmax = 0.9 +/- 0.4 pmol/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 compared 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 results 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.

Animals↗
PubMed2006

Lifelong risk factors for osteoporosis and fractures in elderly women with low body mass index--a population-based study.

Low body weight is associated with an increased risk for osteoporosis and fractures, but the contribution of other lifestyle related factors have not been previously studied within lean elderly women. The present study evaluated the association between lifelong lifestyle factors and bone density, falls and postmenopausal fractures in elderly women with low body mass index (BMI). A population-based sample of 1,222 women aged 70 to 73 years was stratified by BMI tertiles, and all 407 women in the lowest tertile participated. Data on falls and postmenopausal fractures, physical activity, functional capacity, calcium intake, smoking, alcohol intake and medical factors at different ages were obtained by a questionnaire. Calcaneum bone mass as broadband ultrasound attenuation (BUA) was assessed with a quantitative ultrasound (QUS) device, and bone mineral density (BMD) at the distal radius was measured with a dual-energy X-ray absorptiometry (DXA). Low current physical activity was associated with lower calcaneum BUA and factors associated with higher BUA were body weight, low lifetime occupational physical activity, hormone replacement and type 2 diabetes. Weight, type 2 diabetes and thiatzide use were associated with higher radius BMD. The final multivariate model consisted of four independent factors associated with fractures: low lifetime habitual physical activity (OR 3.7, 95% CI 1.9-7.1), diabetes (OR 0.2, 95% CI 0.1-1.0), living alone (OR 1.7, 95% CI 1.0-3.0) and calcaneum BUA (1.8, 95% CI 1.3-2.4). Poor functional ability and symptoms of depression were associated with recent falling. In elderly women with low BMI, lifelong physical activity may protect from fractures, while low calcaneum bone mass and living unpartnered appear to be associated with an increased risk for fractures. Poor functional ability and presence of depression may be associated with risk of falling. Type 2 diabetes may modify the risk of low bone mass and low-trauma postmenopausal fractures. Albeit that the results of this study need to be confirmed in prospective follow-up studies, multifactorial program with the emphasis on physical and social activation in the primary care setting for preventing falls and fractures in lean elderly women is recommended.

Absorptiometry, Photon↗
PubMed1996

Localization of the thiazide sensitive Na-Cl cotransporter, rTSC1 in the rat kidney.

A thiazide sensitive Na-Cl cotransporter, rTSC1, has recently been cloned from a rat kidney cortex cDNA library. The molecular regulation and nephron localization of this protein is unknown. The purpose of this study was to examine the nephron distribution and subcellular localization of the rTSC1 protein in the rat kidney. In situ hybridization showed rTSC1 transcripts were localized to short, convoluted tubule segments in the kidney cortex. Polyclonal antibodies raised against a 110 amino acid segment from the amino terminus of rTSC1 recognized three major bands of 135, 140 and 155 kDa on Western blotting of membrane protein from cortex but not outer medulla of the rat kidney. Immunofluorescence studies using the antibody alone and in double labeling experiments with antibodies against the H+ ATPase and calbindin D28, showed intense labeling of apical membranes in the distal nephron beginning in the initial distal convoluted tubule and terminating within the connecting tubule. The intensity of labeling diminished from proximal to distal sites along the distal tubule. Ultrastructural studies by immunoelectron microscopy showed the cotransporter protein to be localized predominately on apical microvilli of the distal convoluted tubule cells. These results are consistent with rTSC1 encoding the apical thiazide sensitive Na-Cl cotransporter in the distal tubule.

Animals↗
PubMed1996

Thiazide treatment of rats provokes apoptosis in distal tubule cells.

We studied the effects of inhibition of apical NaCl entry on the structural correlates for electrolyte transport in the distal convoluted tubule (DCT) of rats. Thiazide diuretics were used to block NaCl entry specifically in the DCT. Metolazone or hydrochlorothiazide (HCTZ) were applied for three days subcutaneously via osmotic minipumps. The renal epithelial structure of control and treated rats was studied by light and electron microscopy. Distribution of the thiazide-sensitive NaCl cotransporter (rTSC1), calbindin D28K and Ca(2+)-Mg(2+)-ATPase was examined by immunohistochemistry, and the content of rTSC1 transcripts by Northern blot and in situ hybridization. In treated rats the DCT epithelium had lost the structural characteristics of electrolyte transporting epithelia and the cells were in different stages of apoptosis. In damaged cells calbindin D28K and Ca(2+)-Mg(2+)-ATPase were strongly decreased; the rTSC1 was shifted from the luminal membrane to the basal cell half and was found additionally in small membrane vesicles in intercellular and peritubular spaces. Transcripts of rTSC1 were drastically reduced in homogenates of kidney cortex and almost absent in damaged DCT cells. All other tubular segments were unaffected by the treatment. Focal inflammatory infiltrates were found to be specifically surrounding DCT profiles. Thus, inhibition by thiazides of apical NaCl entry into DCT cells is associated with apoptosis of DCT cells and focal peritubular inflammation.

Analysis of Variance↗
PubMed1998

Novel mutations in the thiazide-sensitive NaCl cotransporter gene in patients with Gitelman syndrome with predominant localization to the C-terminal domain.

Gitelman syndrome (familial hypokalemia-hypomagnesemia syndrome) is an autosomal recessive inherited renal disorder characterized by defective tubular reabsorption of magnesium and potassium. In this study a group of 18 unrelated and 2 related Gitelman patients, collected from six different countries have been screened for mutations in the human thiazide-sensitive sodium-chloride cotransporter (SLC12A3) gene. Fourteen novel SLC12A3 mutations are presented along with six mutations described earlier, and three neutral polymorphisms. Among the tested patients are two who carry a total of three heterozygous SLC12A3 mutations. Two-thirds of the total number of mutant SLC12A3 alleles are amino acid substitutions. Most SLC12A3 gene mutations, 14 out of a total of 20, are localized at the intracellular carboxy-terminal domain of the NCCT protein. The pathogenicity of individual SLC12A3 mutations is based upon their predicted effect on SLC12A3 protein, and segregation in family members. Evolutionary conservation of substituted amino acid residues and their frequency in control chromosomes is presented. Identical mutations have been found in Gitelman families from different geographical origin, suggesting ancient mutations originating from a common ancestor. As yet, we have not found any evidence for a possible genotype-phenotype correlation.

Amino Acid Sequence↗
PubMed2003

Thiazide-induced hypocalciuria is accompanied by a decreased expression of Ca2+ transport proteins in kidney.

INTRODUCTION: Thiazide diuretics have the unique characteristic of increasing renal Na+ excretion, while decreasing Ca2+ excretion. However, the molecular mechanism responsible for this thiazide-induced hypocalciuria remains unclear. The present study investigates the effect of thiazides on the expression of the proteins involved in active Ca2+ transport as well as the role of extracellular volume (ECV) status. METHODS: Hydrochlorothiazide (HCTZ), 12 mg/24 hours, was administered during 7 days to Wistar rats by osmotic minipumps. In addition, ECV contraction was either prevented by Na+ repletion or induced by a low-salt diet. Expression levels of the proteins involved in active Ca2+ transport [i.e., epithelial Ca2+ channel (TRPV5/ECaC1), calbindin-D28K, Na+/Ca2+ exchanger (NCX1)], as well as the thiazide-sensitive Na+ Cl- cotransporter (NCC) were determined by real-time quantitative polymerase chain reaction (PCR) and semiquantitative immunohistochemistry. RESULTS: HCTZ significantly reduced urinary Ca2+ excretion (22%+/- 5% relative to controls). Hematocrit was significantly increased, confirming ECV contraction. In addition, Na+ depletion virtually abolished Ca2+ excretion (8%+/- 1%), while Na+ repletion during HCTZ treatment prevented both ECV contraction and hypocalciuria. HCTZ significantly decreased mRNA expression of TRPV5 (71%+/- 6%), calbindin-D28K (53%+/- 6%), NCX1 (51%+/- 8%) and NCC (50%+/- 11%), regardless of ECV status or calciuresis. Immunohistochemistry revealed reduced TRPV5 (43%+/- 2%), calbindin-D28K (59%+/- 1%) and NCC (56%+/- 4%) abundance. Furthermore, during HCTZ treatment, the subset of tubules coexpressing NCC and calbindin-D28K was significantly reduced (43%+/- 5%) and a disturbed cellular localization of NCC was observed. CONCLUSION: These data suggest that ECV contraction is a critical determinant of the thiazide-induced hypocalciuria, which is accompanied by a decreased expression of Ca2+ transport proteins.

Animals↗
PubMed2003

[Gitelman's syndrome: an important differential diagnosis of hypokalemia].

HISTORY AND CLINICAL FINDINGS: A 26-year-old woman presented with fatigue, muscle cramps and weakness. Since the age of 8 years she had moderate hypokalemia of unknown origin that was confirmed on multiple occasions. There was no family history of disease. INVESTIGATIONS: Laboratory tests showed moderate to severe hypokalemia with a serum potassium concentration of 2.7 to 3.0 mmol/l, hypomagnesemia, metabolic alkalosis and pronounced stimulation of the renin-angiotensin-aldosterone system. Despite normal serum calcium levels, urinary calcium excretion was below the detection threshold. Increased natriuresis was observed after administration of furosemide, but not after administration of hydrochlorothiazide. This finding pointed to the presence of a non-functional thiazide-sensitive sodium/chloride cotransporter in the distal convoluted tubule, characteristic for Gitelman's syndrome. Genetic analysis confirmed the diagnosis of Gitelman's syndrome and documented two heterozygous mutations in the gene encoding the sodium/chloride cotransporter. TREATMENT AND COURSE: The patient was treated with 160 mmol potassium and 30 mmol magnesium supplementation per day. Serum potassium was normalized and magnesium serum levels increased. Weakness and fatigue improved markedly. CONCLUSION: Gitelman's syndrome is an important differential diagnosis in the evaluation of the normotensive patient with hypokalemia.

Adult↗
PubMed2006

Anomalous levels of Cl- transporters in the hippocampal subiculum from temporal lobe epilepsy patients make GABA excitatory.

The mRNA levels of NKCC1, an inwardly directed Na(+), K(+)-2Cl(-) cotransporter that facilitates the accumulation of intracellular Cl(-), and of KCC2, an outwardly directed K(+)-Cl(-) cotransporter that extrudes Cl(-), were studied in surgically resected brain specimens from drug-resistant temporal lobe (TL) epilepsy (TLE) patients. Quantitative RT-PCR analyses of the mRNAs extracted from the human TLE-associated brain regions revealed an up-regulation of NKCC1 mRNA and a down-regulation of KCC2 mRNA in the hippocampal subiculum, compared with the hippocampus proper or the TL neocortex, suggesting an abnormal transcription of Cl(-) transporters in the TLE subiculum. In parallel experiments, cell membranes isolated from the same TLE-associated brain regions were injected into Xenopus oocytes that rapidly incorporated human GABA(A) receptors into their surface membrane. The GABA currents elicited in oocytes injected with membranes from the subiculum had a more depolarized reversal potential (E(GABA)) compared with the hippocampus proper or the neocortex. The NKCC1 blocker bumetanide or a temperature decrease of 10 degrees C shifted the GABA-current E(GABA) more negative in oocytes injected with membranes from TLE hippocampal subiculum, matching the E(GABA) of TL neocortex-injected oocytes. We conclude that the anomalous expression of both Cl(-) transporters, NKCC1 and KCC2 [corrected] in TLE hippocampal subiculum probably causes altered Cl(-) transport in the "epileptic" neurons, as revealed in the microtransplanted Xenopus oocytes, and renders GABA aberrantly "exciting," a feature that may contribute to the precipitation of epileptic seizures.

Animals↗
PubMed1998

The thiazide-sensitive Na-Cl cotransporter is an aldosterone-induced protein.

Although the collecting duct is regarded as the primary site at which mineralocorticoids regulate renal sodium transport in the kidney, recent evidence points to the distal convoluted tubule as a possible site of mineralocorticoid action. To investigate whether mineralocorticoids regulate the expression of the thiazide-sensitive Na-Cl cotransporter (TSC), the chief apical sodium entry pathway of distal convoluted tubule cells, we prepared an affinity-purified, peptide-directed antibody to TSC. On immunoblots, the antibody recognized a prominent 165-kDa band in membrane fractions from the renal cortex but not from the renal medulla. Immunofluorescence immunocytochemistry showed TSC labeling only in distal convoluted tubule cells. Semiquantitative immunoblotting studies demonstrated a large increase in TSC expression in the renal cortex of rats on a low-NaCl diet (207 +/- 21% of control diet). Immunofluorescence localization in tissue sections confirmed the strong increase in TSC expression. Treatment of rats for 10 days with a continuous subcutaneous infusion of aldosterone also increased TSC expression (380 +/- 58% of controls). Furthermore, 7-day treatment of rats with an orally administered mineralocorticoid, fludrocortisone, increased TSC expression (656 +/- 114% of controls). We conclude that the distal convoluted tubule is an important site of action of the mineralocorticoid aldosterone, which strongly up-regulates the expression of TSC.

Aldosterone↗
PubMed2004

A single nucleotide polymorphism alters the activity of the renal Na+:Cl- cotransporter and reveals a role for transmembrane segment 4 in chloride and thiazide affinity.

The thiazide-sensitive Na+:Cl- cotransporter is the major salt transport pathway in the distal convoluted tubule of the kidney, and a role of this cotransporter in blood pressure homeostasis has been defined by physiological studies on pressure natriuresis and by its involvement in monogenic diseases that feature arterial hypotension or hypertension. Data base analysis revealed that 135 single nucleotide polymorphisms along the human SLC12A3 gene that encodes the Na+:Cl- cotransporter have been reported. Eight are located within the coding region, and one results in a single amino acid change; the residue glycine at the position 264 is changed to alanine (G264A). This residue is located within the fourth transmembrane domain of the predicted structure. Because Gly-264 is a highly conserved residue, we studied the functional properties of this polymorphism by using in vitro mutagenesis and the heterologous expression system in Xenopus laevis oocytes. G264A resulted in a significant and reproducible reduction ( approximately 50%) in (22)Na+ uptake when compared with the wild type cotransporter. The affinity for extracellular Cl- and for thiazide diuretics was increased in G264A. Western blot analysis showed similar immunoreactive bands between the wild type and the G264A cotransporters, and confocal images of oocytes injected with enhanced green fluorescent protein-tagged wild type and G264A cotransporter showed no differences in the protein surface expression level. These observations suggest that the G264A polymorphism is associated with reduction in the substrate translocation rate of the cotransporter, due to a decrease in the intrinsic activity. Our study also reveals a role of the transmembrane segment 4 in defining the affinity for extracellular Cl- and thiazide diuretics.

Adult↗
PubMed2004

G2736A polymorphism of thiazide-sensitive Na-Cl cotransporter gene predisposes to hypertension in young women.

OBJECTIVE: The thiazide-sensitive Na-Cl cotransporter (TSC) is located in the distal renal tubules. Several mutations of the TSC gene cause Gitelman's syndrome, which is an autosomal recessive disease characterized by low blood pressure and hypokalemia. Recently, an association between TSC gene polymorphisms (Arg904Gln, G2736A; Thr465Thr, C1420T; Gly264Ala, G816C) and essential hypertension has been reported in Sweden. We examined the genetic involvement of the TSC gene in essential hypertension in Japanese. DESIGN: Participants were recruited from outpatients of Osaka University Hospital. We investigated 386 hypertensive and 371 normotensive subjects. METHODS: Genotypes of TSC polymorphisms (G2736A, C1420T, G816C) were determined by the TaqMan polymerase chain reaction (PCR) method, and statistical significance was examined using JMP 5.0.1J (SAS Institute Inc., Cary, North Carolina, USA). The allele frequency of A2736 and T1420 was 6.0 and 3.0%, respectively, whereas we could not detect the G816C polymorphism in this study. Only the G2736A polymorphism was significantly associated with the prevalence of hypertension (P <0.04), and the estimated odds ratio was 1.8 (95% confidence interval, 1.1-3.0) in A2736 allele carriers. The odds ratio for hypertension in A2736 carriers was increased to 2.2 (1.1-4.9) in women (n=413), and further to 3.3 (1.4-8.0) in women with early onset of hypertension (< or = 50 years old). In addition, all subjects with the homozygous A2736 allele in this study (n=2) and the Swedish study (n=5) were hypertensive. CONCLUSION: G2736A polymorphism of the TSC gene is a genetic predisposing factor for essential hypertension in Japanese women.

Age Distribution↗
PubMed1997

Expression of the sodium-chloride cotransporter in osteoblast-like cells: effect of thiazide diuretics.

The use of thiazide diuretics is associated with increased bone mineral density and, in some studies with reduced incidence of fractures, suggesting a potential role for these drugs in the treatment of osteoporosis. Our objective was to examine the effects of thiazides on osteoblast-like cells using the rat UMR-106 osteosarcoma cell line. Treatment of UMR-106 cells with chlorothiazide caused membrane depolarization and a rise of intracellular calcium but had no effect on adenosine 3,5'-cyclic monophosphate accumulation. The rise of intracellular calcium was partially inhibited by nifedipine and removal of extracellular calcium, indicating calcium uptake from the extracellular media, as well as by thapsigargin or dantrolene, indicating contributions from calcium release from intracellular stores. Reverse transcriptase-polymerase chain reaction was used to isolate a partial cDNA clone for the thiazide-sensitive sodium-chloride cotransporter from UMR-106 cells that hybridized to 5.0- and 11.0-kilobase mRNAs when Northern blot analysis was conducted. Antisense oligonucleotides to the sodium-chloride cotransporter specifically inhibited the chlorothiazide-induced depolarization and rise of intracellular calcium and reduced immunofluorescence staining for the sodium-chloride cotransporter protein in UMR-106 cells. We conclude that thiazide diuretics inhibit sodium-chloride cotransporter activity in UMR-106 cells, thereby altering intracellular calcium regulation. These results provide evidence for direct effects of thiazide diuretics on bone cells.

Animals↗
PubMed2004

Ion and diuretic specificity of chimeric proteins between apical Na(+)-K(+)-2Cl(-) and Na(+)-Cl(-) cotransporters.

The mammalian kidney bumetanide-sensitive Na(+)-K(+)-2Cl(-) and thiazide-sensitive Na(+)-Cl(-) cotransporters are the major pathways for salt reabsorption in the thick ascending limb of Henle's loop and distal convoluted tubule, respectively. These cotransporters serve as receptors for the loop- and thiazide-type diuretics, and inactivating mutations of corresponding genes are associated with development of Bartter's syndrome type I and Gitleman's disease, respectively. Structural requirements for ion translocation and diuretic binding specificity are unknown. As an initial approach for analyzing structural determinants conferring ion or diuretic preferences in these cotransporters, we exploited functional differences and structural similarities between Na(+)-K(+)-2Cl(-) and Na(+)-Cl(-) cotransporters to design and study chimeric proteins in which the NH(2)-terminal and/or COOH-terminal domains were switched between each other. Thus six chimeric proteins were produced. Using the heterologous expression system of Xenopus laevis oocytes, we observed that four chimeras exhibited functional activity. Our results revealed that, in the Na(+)-K(+)-2Cl(-) cotransporter, ion translocation and diuretic binding specificity are determined by the central hydrophobic domain. Thus NH(2)-terminal and COOH-terminal domains do not play a role in defining these properties. A similar conclusion can be suggested for the Na(+)-Cl(-) cotransporter.

Animals↗
PubMed1992

Luminal influences on potassium secretion: chloride, sodium, and thiazide diuretics.

In the presence of Cl-, K+ secretion by the distal tubule saturates with increasing luminal Na+ concentration. Apparent maximal K+ secretion is attained with luminal Na+ concentrations of 40 mM. The results of the present study show that lowering the Cl- concentration of luminal fluid can increase the level of Na(+)-stimulated K+ secretion beyond the maximal level attained in the presence of Cl-. The effect of lowering luminal Cl- concentration to less than 10 mM on K+ secretion is greater with higher Na+ concentration. Under these conditions, chlorothiazide decreases K+ secretion. When chlorothiazide is present, changing the Na+ concentration does not affect K+ secretion. Because in rats a thiazide effect is attributed primarily to the distal convoluted tubule (DCT), we postulate that it is primarily DCT cells that increase K+ secretion when Na+ concentration is raised in the presence of low luminal Cl- concentration. We propose that the rat DCT cells have both an absorptive Na(+)-Cl- cotransport mechanism and a secretory K(+)-Cl- cotransport mechanism in the luminal membrane that can mediate the apparent exchange of Na+ for K+.

Animals↗
PubMed1993

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.

Animals↗