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Molecular biology of distal nephron sodium transport mechanisms

1999; Elsevier BV; Volume: 56; Issue: 4 Linguagem: Inglês

10.1046/j.1523-1755.1999.00712.x

1523-1755

Gerardo Gamba,

Ion channel regulation and function

A 54-year-old man was admitted to the emergency room because he was confused and had somnolence alternating with periods of muscular rigidity. Three weeks before the admission, progressive and generalized muscular weakness had developed. On physical examination he was disoriented and had hyperreflexia and spontaneous rotatory eye movements. His blood pressure was 158/88 mm Hg. Laboratory analysis disclosed: serum glucose, 150 mg/dl; sodium, 116 mEq/liter; chloride, 81 mEq/liter; potassium, 2.9 mEq/liter; bicarbonate, 26.8 mmol/liter; arterial pH, 7.47; and PCO2, 36.2 mm Hg. Cerebral magnetic resonance imaging (MRI) showed no focal lesions. The patient had a history of non-insulin-dependent diabetes mellitus for several years with progressive retinopathy and nephropathy. He had been diagnosed one month before this admission with advanced nephropathy characterized by generalized severe edema, mild chronic renal failure, and arterial hypertension. Physical examination confirmed the presence of edema. Arterial pressure at that time was 207/105 mm Hg. Laboratory analysis then revealed: serum glucose, 211 mg/dl; serum creatinine, 2.7 mg/dl; BUN, 37 mg/dl; cholesterol, 364 mg/dl; albumin, 3.5 g/dl; calcium, 9 mg/dl; phosphorus, 6.3 mg/dl; sodium, 140 mEq/liter; and potassium 4.0 mEq/liter. The 24-hour urinary protein excretion was 4.6 g. The patient initially was treated for edema with furosemide, 40 mg/day, and hydrochlorothiazide, 12.5 mg/day. Because the edema did not respond to treatment, the furosemide dose was progressively increased over the following two weeks to 200 mg/day; the thiazide dose remained the same. Because of the finding of hyponatremia, diuretics were withdrawn after his admission to the emergency room. The patient was treated with water restriction and oral sodium repletion; his confusion and somnolence gradually resolved over the first five days in hospital. He left the hospital 10 days after admission with normal serum electrolytes and arterial blood gases. DR. GERARDO GAMBA (Chief, Molecular Physiology Unit, Instituto Nacional de la Nutrición Salvador Zubirán, and Instituto de Investigationes Biomédicas, Universidad Nacional Autónoma de México, Mexico City, Mexico): The fine control of sodium (Na+) excretion and urine concentration takes place in the distal nephron. As discussed by Rose in a previous Forum[1.Rose B.D. Diuretics.Kidney Int. 1991; 39: 336-352Abstract Full Text PDF PubMed Scopus (128) Google Scholar], the major Na+ transport mechanisms of the distal nephron (defined as that portion of the nephron starting with the thick ascending loop) also serve as targets for diuretic action. The patient presented here had a clinical history that is not uncommon in clinical practice. He was admitted to the emergency room with progressive muscular weakness that evolved to somnolence and a confusional state. Magnetic resonance imaging revealed no evidence of cerebral lesions, and his clinical status was explained by metabolic derangements such as severe hyponatremia, hypokalemia, and metabolic alkalosis. These metabolic and clinical alterations resulted from inhibition of two major sodium transport proteins of the distal nephron. The patient had been treated for several weeks before admission to the emergency room with a combination of two diuretic drugs: furosemide and hydrochlorothiazide. The first agent inhibits the function of the Na+:K+:2Cl- cotransporter in the thick ascending limb (TAL), and the second one inhibits the function of the Na+:Cl- cotransporter in the distal convoluted tubule (DCT). Inhibition of sodium reabsorption in two contiguous nephron segments resulted in potentiation of the diuretic action. In addition, although the patient had moderate chronic renal failure, his exaggerated response to diuretics resulted in renal loss of sodium, potassium, and hydrogen. Sodium chloride and potassium repletion plus withdrawal of the diuretics restored the metabolic state of the patient. The general mechanism of sodium reabsorption along the nephron is based on the following physiologic events: The Na+:K+:ATPase polarized to the basolateral membrane provides the electrochemical gradient that allows sodium to be transported from the lumen to the interstitial space; in the apical membrane, with the exception of the collecting duct (CD), Na+ is cotransported or contra-transported with other molecules (for example, glucose, amino acid residues) or ions (for example, Cl-, K+), which are carried against their gradient by a group of proteins collectively known as secondary transporters. The presence of each particular type of secondary transporter varies along the nephron in such a way that part of the characteristic heterogeneity of the nephron is explained by the amount and type of transporters expressed in each segment. For instance, the Na+:glucose cotransporter is expressed only in the proximal tubule. Thus, this is the only segment of the nephron in which glucose is reabsorbed. In the CD, and probably at the end of the distal tubule[2.Costanzo L.S. Comparison of calcium and sodium transport in early and late rat distal tubules: Effect of amiloride.Am J Physiol. 1984; 246: F937-F945PubMed Google Scholar], Na+ delivery is lower than in the rest of the nephron, especially during sodium depletion; this decrease reduces the possibility for Na+ to be carried by cotransporters. In the CD, Na+ transport occurs electrogenically in the apical membrane of the principal cells through epithelial sodium-selective channels. In this Forum, I will present an overview of the molecular biology of the distal nephron Na+ transport mechanisms. I will focus on the Na+ pathways in the TAL, DCT, and CD, which are known by many clinicians as the diuretic receptors. The TAL reabsorbs 15% to 20% of the glomerular filtrate, regulates divalent mineral excretion, and plays a key role in the production and maintenance of renal medullary hypertonicity. This last function provides the kidney with the ability to form urine that can be more diluted or concentrated than plasma. This functional capacity is essential for the survival of land mammals, including humans. Figure 1a shows the molecular physiology of sodium reabsorption in the TAL. The major pathway for sodium reabsorption is the apical membrane Na+:K+:2Cl- cotransporter, the main pharmacologic target of loop diuretics (furosemide, bumetanide, ethacrynic acid, torasemide, and piretanide)[3.Hebert S. Nephron heterogeneity.in: Windhager E.E. Handbook of Physiology: Renal Physiology. Oxford University Press, New York1992: 875Google Scholar]. Net NaCl reabsorption in the TAL is increased by hormones generating cAMP via their respective Gs-coupled receptors, such as vasopressin[4.Hebert S.C. Culpepper R.M. Andreoli T.E. NaCl transport in mouse medullary thick ascending limbs. II. ADH enhancement of transcellular NaCl cotransport: Origin of transepithelial voltage.Am J Physiol. 1981; 241: F432-F442PubMed Google Scholar]. In the mouse, vasopressin modulates the NaCl transport mode. In its absence, NaCl is transported in the TAL through a K+-independent, but nevertheless furosemide-sensitive, Na+:Cl- mechanism, whereas its presence switches the NaCl transport mode to the furosemide-sensitive Na+:K+:2Cl- cotransporter[5.Sun A. Grossman E.B. Lombardi M. Hebert S.C. Vasopressin alters the mechanism of apical Cl- entry from Na+:Cl- to Na+:K+:2Cl- cotransport in mouse medullary thick ascending limb.J Membr Biol. 1991; 120: 83-94Crossref PubMed Scopus (87) Google Scholar]. A similar regulatory mechanism occurs in rabbit TAL, which exhibits a furosemide-sensitive Na+:Cl- cotransporter that is switched to a Na+:K+:2Cl- mode in the presence of hypertonicity[6.Eveloff J. Calamia J. Effect of osmolarity on cation fluxes in medullary thick ascending limb cells.Am J Physiol. 1986; 250: F176-F180PubMed Google Scholar]. The presence of two furosemide-sensitive cotransport mechanisms in the TAL is supported by the finding of two classes of bumetanide-binding sites in membrane preparations from whole mouse kidneys[7.Haas M. Dunham P.B. Forbush III, B. [3H]bumetanide binding to mouse kidney membranes: Identification of corresponding membrane protein.Am J Physiol. 1991; 260: C791-C804PubMed Google Scholar], high-affinity sites of approximately 150 kDa and low-affinity sites of approximately 75 kDa. This finding suggests that Na+:Cl- and Na+:K+:2Cl- cotransporters in the TAL could correspond to two different proteins. The switching of transport mode from Na+:Cl- to Na+:K+:2Cl-, together with the activation of the apical inwardly rectifying K+ channel (ROMK), also regulated by vasopressin, is of crucial importance in the TAL function, because K+ entering the cell through the Na+:K+:2Cl- cotransporter is recycled to the lumen by ROMK, generating a lumen-positive potential difference that drives the reabsorption of cations through a paracellular pathway. Thus, vasopressin-induced cAMP generation is a fundamental mechanism for regulating cation transport in the TAL because it doubles Na+ reabsorption without increasing energy output and also because it is responsible for the reabsorption of other cations such as Ca2+ and Mg2+[3.Hebert S. Nephron heterogeneity.in: Windhager E.E. Handbook of Physiology: Renal Physiology. Oxford University Press, New York1992: 875Google Scholar]. Hence, combining loop diuretics with saline loading is the basis for hypercalcemia therapy, as inhibition of the Na+:K+:2Cl- cotransporter increases urinary calcium excretion and reduces plasma calcium concentration. Two genes for the Na+:K+:2Cl- cotransporter have been identified: (1) the renal-specific BSC1/NKCC2, located on human chromosome 15, which encodes the TAL Na+:K+:2Cl- cotransporter (hereafter referred to as BSC1)[8.Gamba G. Miyanoshita A. Lombardi M. Lytton J. Lee W.S. Hediger M.A. Hebert S.C. Molecular cloning, primary structure and characterization of two members of the mammalian electroneutral sodium-(potassium)-chloride cotransporter family expressed in kidney.J Biol Chem. 1994; 296: 17713-17722Google Scholar],[9.Payne J.A. Forbush III, B. Alternatively spliced isoforms of the putative renal Na-K-Cl cotransporter are differentially distributed within the rabbit kidney.Proc Natl Acad Sci USA. 1994; 91: 4544-4548Crossref PubMed Scopus (243) Google Scholar] and (2) the ubiquitous BSC2/NKCC1, located on human chromosome 5q23.3, which is expressed in several tissues and cell lines encoding for the basolateral and non-epithelial Na+:K+:2Cl- cotransporter (hereafter referred to as BSC2)[10.Xu J.-C. Lytle C. Zhu T.T. Payne A.J. Benz Jr, E. Forbush III, B. Molecular cloning and functional expression of the bumetanide-sensitive Na-K-Cl cotransporter.Proc Natl Acad Sci USA. 1994; 91: 2201-2205Crossref PubMed Scopus (355) Google Scholar],[11.Delpire E. Rauchman M.I. Beier D.R. Hebert S.C. Gullans S.R. Molecular cloning and chromosome localization of a putative basolateral Na+-K+-2Cl- cotransporter from mouse inner medullary collecting duct (mIMCD-3) cells.J Biol Chem. 1994; 269: 25677-25683Abstract Full Text PDF PubMed Google Scholar]. Functional expression of BSC1 and BSC2 in Xenopus laevis oocytes and HEK-293 cells, respectively, induces the expected expression of a bumetanide-sensitive86Rb+ transport mechanism (substituting K+) that is Na+ and Cl- dependent[8.Gamba G. Miyanoshita A. Lombardi M. Lytton J. Lee W.S. Hediger M.A. Hebert S.C. Molecular cloning, primary structure and characterization of two members of the mammalian electroneutral sodium-(potassium)-chloride cotransporter family expressed in kidney.J Biol Chem. 1994; 296: 17713-17722Google Scholar],[10.Xu J.-C. Lytle C. Zhu T.T. Payne A.J. Benz Jr, E. Forbush III, B. Molecular cloning and functional expression of the bumetanide-sensitive Na-K-Cl cotransporter.Proc Natl Acad Sci USA. 1994; 91: 2201-2205Crossref PubMed Scopus (355) Google Scholar]. Both BSC1 and BSC2 share a basic structural topology with the rest of the members of the cation-chloride gene family. They are glycoproteins of 1095 to 1212 amino acid residues with molecular weights of approximately 165 kDa and a central hydrophobic region containing 12 putative transmembrane domains flanked by amino- and carboxy-terminal hydrophilic domains located within the cell. The extracellular hydrophilic loop between putative transmembrane domains S7 and S8 contains three N-glycosylation motifs. Several putative PKA and PKC phosphorylation sites are present within the amino- and carboxy-terminal domains Figure 1b. At least six isoforms of BSC1 are expressed in the mouse kidney by the combination of two alternative splicing mechanisms Figure 2[12.Mount D.B. Baekgard A. Hall A.E. Plata C. Xu J. Beier D.R. Gamba G. Hebert S.C. Isoforms of the Na-K-2Cl transporter in murine TAL. I. Molecular characterization and intrarenal localization.Am J Physiol. 1999; 276: F347-F358PubMed Google Scholar]. One is due to the presence of three mutually exclusive cassette exons of 96 bp designated A, B, and F, which encode for 32 amino acid residues corresponding to the putative second transmembrane domain (S2) and the contiguous intracellular loop between S2 and S3[9.Payne J.A. Forbush III, B. Alternatively spliced isoforms of the putative renal Na-K-Cl cotransporter are differentially distributed within the rabbit kidney.Proc Natl Acad Sci USA. 1994; 91: 4544-4548Crossref PubMed Scopus (243) Google Scholar],[13.Igarashi P. Vanden Heuver G.B. Payne J.A. Forbush III, B. Cloning, embryonic expression, and alternative splicing of a murine kidney-specific Na-K-Cl cotransporter.Am J Physiol. 1995; 269: F406-F418Google Scholar]. This splicing produces three BSC1 proteins that are identical, with the exception of the 32 amino acids encoded by A, B, or F cassettes. The second splicing of the BSC1 gene is the utilization of a poly-adenylation site in the intron between coding exons 16 and 17, which predicts a protein with a significantly shorter C-terminal domain. This splicing produces two BSC1 proteins that are identical at the N-terminus and the transmembrane domains, but which differ in the length and sequence of the C-terminal domain. The longer isoforms exhibit a C-terminus of 457 amino acid residues, from which the last 383 are not present in the shorter isoform. In contrast, the shorter, truncated isoforms exhibit a C-terminus of 129 residues, from which the last 55 are not present in the longer isoforms. Interestingly, the long and short C-terminal domains contain different putative protein kinase A (PKA) and protein kinase C (PKC) phosphorylation sites. As Figure 2 shows, we have designated the longer and shorter isoforms as mBSC1-9 and mBSC1-4, respectively. The two splicing events appear to be independent from each other in such a way that a total of six isoforms is produced in the mouse kidney[12.Mount D.B. Baekgard A. Hall A.E. Plata C. Xu J. Beier D.R. Gamba G. Hebert S.C. Isoforms of the Na-K-2Cl transporter in murine TAL. I. Molecular characterization and intrarenal localization.Am J Physiol. 1999; 276: F347-F358PubMed Google Scholar]. The three mBSC1-9 isoforms cRNA (mBSC1-A9, mBSC1-B9, and mBSC1-F9) induce expression of a bumetanide-sensitive Na+:K+:2Cl- cotransporter when injected in Xenopus oocytes[14.Plata C. Mount D.B. Rubio V. Hebert S.C. Gamba G. Isoforms of the Na-K-2Cl cotransporter in murine TAL. II. Functional characterization and activation by cyclic-AMP.Am J Physiol. 1999; 276: F359-F366PubMed Google Scholar]. The functional significance of these isoforms is not yet understood. However, in-situ hybridization[13.Igarashi P. Vanden Heuver G.B. Payne J.A. Forbush III, B. Cloning, embryonic expression, and alternative splicing of a murine kidney-specific Na-K-Cl cotransporter.Am J Physiol. 1995; 269: F406-F418Google Scholar] and single-nephron polymerase chain reaction (PCR) studies[15.Yang T. Huang Y.G. Singh I. Schnermann J. Briggs J.P. Localization of bumetanide- and thiazide-sensitive Na-K-Cl cotransporters along the rat nephron.Am J Physiol. 1996; 271: F931-F939PubMed Google Scholar] using isoform-specific probes and primers, respectively, have shown that the mBSC1-9-type isoforms exhibit differential expression along the TAL. The F-isoform is expressed at the inner stripe of the outer medulla, A-isoform is mainly expressed at the outer stripe of the outer medulla, and B-isoform is exclusively localized at the cortical TAL. This distribution suggests that A, B, and F cassettes could endow different affinity and/or transport capacity to mBSC1-9 isoforms. Unfortunately, kinetic analysis of the three isoforms is not available. In this regard, however, experiments with human and shark basolateral isoforms, BSC2, provide preliminary evidence that kinetic properties of extracellular Na+ and K+, as well as bumetanide inhibition, but not extracellular Cl- concentration, are at least partly determined by the sequence of the first and second membrane domains[16.Insering P. Jacoby S.C. Forbush III, B. The role of transmembrane domain 2 in cation transport by the Na-K-Cl cotransporter.Proc Natl Acad Sci USA. 1998; 95: 7179-7184Crossref PubMed Scopus (71) Google Scholar]. The functional properties of mBSC1-4 isoforms have not yet been established because these isoforms do not express Na+:K+:2Cl- cotransport in Xenopus oocytes. However, recent data from our laboratory show that mBSC1-4 isoforms exert a dominant-negative function on the ion transport expressed by full-length mBSC1-9 isoforms[14.Plata C. Mount D.B. Rubio V. Hebert S.C. Gamba G. Isoforms of the Na-K-2Cl cotransporter in murine TAL. II. Functional characterization and activation by cyclic-AMP.Am J Physiol. 1999; 276: F359-F366PubMed Google Scholar]. On one hand, Xenopus oocytes injected with any of the three mBSC1-9 isoforms showed that PKA activation or inhibition with cAMP or H89, respectively, does not affect the function of the Na+:K+:2Cl- cotransporter[14.Plata C. Mount D.B. Rubio V. Hebert S.C. Gamba G. Isoforms of the Na-K-2Cl cotransporter in murine TAL. II. Functional characterization and activation by cyclic-AMP.Am J Physiol. 1999; 276: F359-F366PubMed Google Scholar]. On the other hand, the dominant-negative effect of mBSC1-4 is reversed by cAMP. Therefore it is highly likely that the negative effect of mBSC1-4 plays a key role in the cAMP-induced activation of the Na+:K+:2Cl- cotransporter. Supporting this conclusion, Mount et al, using immunofluorescence studies, have recently shown that both isoforms are coexpressed in TAL cells. The mBSC1-9-specific antibodies label the apical membrane of all cells along the entire length of TAL, whereas mBSC1-4 expression exhibits a heterogeneous pattern in which not all TAL cells are labeled. In addition, cortical TAL appears to express less mBSC1-4 than do outer medullary segments. This heterogeneity may underlie the observed difference in vasopressin-sensitive Na+:Cl- cotransport in cortical TAL versus medullary TAL[17.Hebert S.C. Culpepper R.M. Andreoli T.E. NaCl transport in mouse medullary thick ascending limbs. I. Functional nephron heterogeneity and ADH-stimulated NaCl cotransport.Am J Physiol. 1981; 241: F412-F431PubMed Google Scholar]. Molecular probes of BSC1 and polyclonal antibodies directed against BSC1 have been used in recent years to determine the role of BSC1 gene expression under several physiologic and pathophysiologic conditions. We have shown that high or low NaCl intake, dehydration, water loading, and furosemide administration do not affect BSC1 expression at the mRNA level[18.Moreno G. Merino A. Mercado A. Herrera J.P. González-Salazar J. Correa-Rotter R. Hebert S.C. Gamba G. Electroneutral Na-coupled cotransporter expression in the kidney during variations of NaCl and water metabolism.Hypertension. 1998; 31: 1002-1006Crossref PubMed Scopus (34) Google Scholar]. In contrast, modifications in K+ and acid-base metabolism seem to affect BSC1 gene expression. It has been known for many years that severe potassium depletion increases urinary chloride excretion[19.Garella S. Chazan J. Cohen J.J. Saline-resistant metabolic alkalosis or "chloride wasting nephropathy".Ann Intern Med. 1970; 73: 31-35Crossref PubMed Scopus (50) Google Scholar] at least in part due to reduction in Cl- reabsorption in TAL and DCT[20.Luke R.G. Wright F.S. Fowler N. Kashgarian M. Giebisch G.H. Effect of potassium depletion on renal tubule chloride transport.Kidney Int. 1978; 14: 414-427Abstract Full Text PDF PubMed Scopus (37) Google Scholar]. In a recent study, Amlal et al observed that potassium depletion reduced Cl- reabsorption in the TAL and downregulated BSC1 mRNA expression[21.Amlal H. Wang Z. Soleimani M. Potassium depletion downregulates chloride-absorbing transporters in rat kidney.J Clin Invest. 1998; 101: 1045-1054Crossref PubMed Scopus (51) Google Scholar]. The Na+:K+:2Cl- cotransporter also is involved in renal acid excretion because NH4+ can substitute for K+ yielding a Na+:NH4+:2Cl- cotransporter. Through this mechanism, the NH4+ produced in the proximal tubule usually is reabsorbed in the TAL and recirculated into the medulla to be excreted by the collecting duct. Thus, one of the potential responses to acidosis is increased reabsorption of NH4+ by the TAL. This hypothesis is supported by the fact that acidosis increases mRNA and transport activity of BSC1[22.Attmane-Elakeb A. Mount D.B. Sibella V. Vernimmen C. Hebert S.C. Bichara M. Stimulation by in vivo and in vitro metabolic acidosis of expression of rBSC-1, the Na+-K+ (NH4+)-2Cl- cotransporter of the rat medullary thick ascending limb.J Biol Chem. 1998; 273: 33681-33691Crossref PubMed Scopus (58) Google Scholar]. Finally, downregulation of BSC1 protein expression in the kidney from rats with adriamycin-induced nephrotic syndrome[23.Fernández-Llama P. Andrews P. Ecelbarger C.A. Nielsen S. Knepper M.A. Concentrating defect in experimental nephrotic syndrome: Altered expression of aquaporins and thick ascending limb Na+ transporters.Kidney Int. 1998; 54: 170-179Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar] might explain the defect in the diluting capacity observed in the nephrotic syndrome. Mutations of the human BSC1 gene (S1c12a1)[24.Simon D.B. Karet F.E. Hamdan J.M. Dipietro A. Sanjad S.A. Lifton R.P. Bartter's syndrome, hypokalaemic alkalosis with hypercalciuria, is caused by mutations in the Na-K-2Cl cotransporter NKCC2.Nat Genet. 1996; 13: 183-188Crossref PubMed Scopus (753) Google Scholar],[25.Vargas-Poussou R. Feldman D. Vollmer M. Konrad M. Kelly L. Van Der Heuvel LPWJ Tebouri L. Brandis M. Karolyi L. Hebert S.C. Lemmink H.H. Deschênes G. Hildebrandt F. Seyberth H.W. Guay-Woodford L.M. Knoers NVAM Antignac C. Novel molecular variants of the Na-K-2Cl cotransporter gene are responsible for antenatal Bartter syndrome.Am J Hum Genet. 1998; 62: 1332-1340Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar] occur in patients with classic and antenatal Bartter's syndrome, a monogenic disease characterized by metabolic alkalosis, hypokalemia, severe salt wasting, and hypercalciuria. Mutations range from alterations on a single amino acid residue to premature termination of the cotransporter protein. No functional studies of mutated clones have been reported, however. Bartter's syndrome is a monogenic but heterogeneous disease; three genes have been identified in the TAL as causes of the syndrome: BSC1[24.Simon D.B. Karet F.E. Hamdan J.M. Dipietro A. Sanjad S.A. Lifton R.P. Bartter's syndrome, hypokalaemic alkalosis with hypercalciuria, is caused by mutations in the Na-K-2Cl cotransporter NKCC2.Nat Genet. 1996; 13: 183-188Crossref PubMed Scopus (753) Google Scholar],[25.Vargas-Poussou R. Feldman D. Vollmer M. Konrad M. Kelly L. Van Der Heuvel LPWJ Tebouri L. Brandis M. Karolyi L. Hebert S.C. Lemmink H.H. Deschênes G. Hildebrandt F. Seyberth H.W. Guay-Woodford L.M. Knoers NVAM Antignac C. Novel molecular variants of the Na-K-2Cl cotransporter gene are responsible for antenatal Bartter syndrome.Am J Hum Genet. 1998; 62: 1332-1340Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar], the apical ATP-sensitive K+ channel[26.Simon D.B. Karet F.E. Rodriguez-Soriano J. Hamdan J.H. Dipietro A. Trachtman H. Sanjad S.A. Lifton R.P. Genetic heterogeneity of Bartter's syndrome revealed by mutations in the K+ channel, ROMK.Nat Genet. 1996; 14: 152-156Crossref PubMed Scopus (688) Google Scholar], and the basolateral Cl- channel[27.Simon D.B. Bindra R. Mansfield T.A. Nelson-Williams C. Mendonca E. Stone R. Schurman S. Nayir A. Alpay H. Bakkaloglu A. Rodriguez-Soriano J. Morales J.M. Sanjad S.A. Taylor C.M. Pilz D. Brem A. Trachtman H. Griswold W. Richard G.A. John E. Lifton R.P. Mutations in the chloride channel gene, CLCNKB, cause Bartter's syndrome type III.Nat Genet. 1997; 17: 171-178Crossref PubMed Scopus (728) Google Scholar]. In addition, some of the kindreds studied by Simon et al have no evidence of mutations in any of these three genes[27.Simon D.B. Bindra R. Mansfield T.A. Nelson-Williams C. Mendonca E. Stone R. Schurman S. Nayir A. Alpay H. Bakkaloglu A. Rodriguez-Soriano J. Morales J.M. Sanjad S.A. Taylor C.M. Pilz D. Brem A. Trachtman H. Griswold W. Richard G.A. John E. Lifton R.P. Mutations in the chloride channel gene, CLCNKB, cause Bartter's syndrome type III.Nat Genet. 1997; 17: 171-178Crossref PubMed Scopus (728) Google Scholar], so at least a fourth gene must be implicated. Kurtz discussed the clinical, physiologic, and genetic alterations in Bartter's syndrome in detail in a recent Forum[28.Kurtz I. Molecular pathogenesis of Bartter's and Gitelman's syndromes.Kidney Int. 1998; 54: 1396-1410Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar]. The distal convoluted tubule (DCT) reabsorbs 5% to 7% of the glomerular filtrate. Figure 3a shows the molecular physiology of Na+ reabsorption in the DCT. The activity of the Na+:K+:ATPase in DCT is comparable to that in the TAL[29.Doucet A. Function and control of Na-K-ATPase in single nephron segments of the mammalian kidney.Kidney Int. 1988; 34: 749-760Abstract Full Text PDF PubMed Scopus (88) Google Scholar]. The major sodium reabsorption pathway in the apical membrane is the Na+:Cl- cotransporter, which is the major target for benzothiadiazine-type diuretics (chlorthalidone, hydrochlorothiazide, bendroflumethiazide, metolazone)[30.Ellison D.H. Velazquez H. Wright F.S. Thiazide-sensitive sodium chloride cotransport in early distal tubule.Am J Physiol. 1987; 253: F546-F554PubMed Google Scholar]. Loop diuretics do not inhibit this cotransporter. In addition to Na+ transport, the DCT is involved in calcium excretion and in urine concentration just as is the TAL[31.Hebert S.C. Roles of Na-K-2Cl and Na-Cl cotransporters and ROMK potassium channels in urinary concentrating mechanism.Am J Physiol. 1998; 275: F325-F327PubMed Google Scholar]. Sodium and Ca2+ reabsorption in the DCT are inversely related. Thus, inhibition of the Na+:Cl- cotransporter with thiazide diuretics increases Ca2+ reabsorption. How thiazides affect calcium reabsorption is still unclear, but they might be functionally linked through an indirect mechanism. Thiazides inhibit NaCl entry at the apical membrane, whereas intracellular Na+ continuously leaves the cells through the Na+:K+:ATPase at the basolateral membrane. This extrusion of Na+ reduces the intracellular Na+ concentration, thereby hyperpolarizing DCT cells and stimulating Ca2+ entry at the apical membrane through Ca2+ channels[32.Gesek F.A. Friedman P.A. Mechanism of calcium transport stimulated by chlorothiazide in mouse distal convoluted tubule cells.J Clin Invest. 1992; 90: 429-438Crossref PubMed Scopus (163) Google Scholar]. This secondary effect of thiazides on Ca2+ reabsorption constitutes the basis for their use in the treatment of calcium stone disease and also might explain the protective effect of thiazides in osteoporosis[33.Ray W.A. Griffin M.R. Downey W. Melton III, Lj Long-term use of thiazide diuretics and risk of hip fracture.Lancet. 1989; 1: 687-690Abstract PubMed Scopus (186) Google Scholar],[34.Wasnich R. Davis J. Ross P. Vogel J. Effect of thiazide on rates of bone mineral loss: A longitudinal study.Br Med J. 1990; 301: 1303-1305Crossref PubMed Scopus (92) Google Scholar]. The thiazide-sensitive Na+:Cl- cotransporter from the winter flounder urinary bladder was the first electroneutral Na+-coupled Cl- cotransporter to be identified at the molecular level following a functional expression strategy in Xenopus laevis oocytes (flTSC)[35.Gamba G. Saltzberg S.N. Lombardi M. Miyanoshita A. Lytton J. Hediger M.A. Brenner B.M. Hebert S.C. Primary structure and functional expression of a cDNA encoding the thiazide-sensitive, electroneutral sodium-chloride cotransporter.Proc Natl Acad Sci USA. 1993; 90: 2749-2753Crossref PubMed Scopus (322) Google Scholar]. Then an RNA probe constructed from the fish cDNA was used to clone, by homology, thiazide-sensitive cotransporter (TSC) from rat kidney (rTSC)[8.Gamba G. Miyanoshita A. Lombardi M. Lytton J. Lee W.S. Hediger M.A. Hebert S.C. Molecular cloning, primary structure and characterization of two members of the mammalian electroneutral sodium-(potassium)-chloride cotransporter family expressed in kidney.J Biol Chem. 1994; 296: 17713-17722Google Scholar]. Functional expression of flTSC and rTSC in Xenopus oocytes gives rise to a22Na+ transport uptake mechanism that is Cl--dependent and inhibitable by thiazide-type diuretics, with an inhibition profile (polythiazide > metolazone > hydrochlorothiazide > chlorothiazide) similar to that previously shown for inhibition of Cl--dependent Na+ absorption[36.Li J.H. Zuzack J.S. Kau S.T. Winter flounder urinary bladder as a model tissue for assessing the potency of thiazide diuretics,.Diuretics III: Chemistry, Pharmacology, and Clinical Applications. edited by PUSCHETT JB, GREENBERG A. Elsevier,