The NaCl Cotransporter Gene and Protein

Major advances have been made over the past few years in our understanding of the actions of thiazide diuretics and this is due in large part to the molecular identification of a family of cation/chloride cotransporters to which the Na/Cl transporter belongs (see Table 2). This information has enhanced our thinking about these transporter proteins. It is now clear that Na/Cl cotransport activity represents the function of a single membrane protein belonging to the newly identified family of proteins, the electroneutral cation/chloride cotransporters.

Cotransporter Gene (Slcl2a3) and Tissue Expression

A single distinct Na/Cl cotransporter gene (Slcl2a3) has been cloned from mammalian tissue (see Table 2). Transcripts encoding the Na/Cl cotransporter are expressed predominantly in the kidney. Extrarenal expression has been shown in osteoblast-like cells and preliminary observations have suggested expression in some other cells (e.g., pancreatic /? cells and testis). The overall extent of extrarenal expression of the Na/Cl cotransporter is currently under intense investigation but potential roles for Na/Cl cotransport in nonrenal tissues in the systemic actions or adverse effects of thiazide diuretics remains speculative. In the kidney, Na/Cl cotransporter expression is restricted to distal convoluted tubule cells in the rat and rabbit kidney. Polyclonal antibodies raised against the rat Na/Cl cotransporter show that the protein is normally limited to the apical surface of distal convoluted tubule cells. Electron micrographs reveal cotransporter protein on the short microplicae and in subapical vesicles. It is not known whether the subapical vesicles represent a pool of co-transporter that can cycle into the membrane and become functional in NaCl transport or are merely part of the protein degradation pathway. Interestingly, this Na/Cl cotransporter antibody was used in a recent study by Kaissling and co-workers showing that thiazide diuretic treatment of rats provoked apoptosis of distal tubule cells. In the rat, Na/Cl cotransport is the sole mechanism of Na+ entry in distal convoluted tubule cells. It is possible that this may represent a part of remodeling of the distal convoluted tubule epithelium when NaCl entry into cells is dramatically reduced (e.g., with thiazide diuretics). However, more work is needed to examine this hypothesis and to determine if the same effect obtains in other species, such as mice, where Na+ entry is mediated both by Na/Cl cotransport and by apical membrane amiloride-sensitive Na+ channels (see below).

Ion transport function and diuretic sensitivities of the rat Na/Cl cotransporter, TSC1, were characterized in Xenopus laevis frog oocytes injected with synthetic messenger RNA made from cotransporter complementary DNA. Co-transporter RNA-injected oocytes exhibited high levels of chloride-dependent tracer sodium uptake. This sodium uptake was specifically inhibited by thiazides such as hydrochlorothiazide (Diuril) or metolazone (Zaroxolyn), whereas the loop diuretic bumetanide had no effect. Thus Slcl2a3 (TSC1) is predominantly expressed in the mammalian kidney and encodes the thiazide diuretic-sensitive Na/Cl cotransporter protein that mediates NaCl absorption in the distal convoluted tubule.

Cotransporter Stoichiometry, Topology, and Diuretic Interactions

The proposed order of ion binding to the thiazide-sensitive Na/Cl cotransporter is shown in Fig. 12A. For the rat distal convoluted tubule a stoichiometry of lNa+: 1C1" for the cotransporter was established by in vivo perfusion. Sodium binds first and then CI" (Na+ —> CI"). Thiazide-like diuretics such as metolazone (Zaroxolyn) appear to bind at or near the CI" site on the transporter protein in a competitive fashion.

The Na/Cl cotransporter is a relatively large protein with a core molecular weight of about 110 kDa. This thiazide-sensitive Na/Cl cotransporter protein has an overall topology (Fig. 12B) that is similar to the Na/K/2C1 cotransport-ers (Fig. 8B) with a large hydrophobic central region of many (possibly 12) membrane-crossing helices flanked by large hydrophilic regions that appear to face the interior of the cell. At least two sugar residues are linked to an extracellular loop between the 7th and 8th membrane-spanning segment, making this cotransporter a glycoprotein and increasing its apparent molecular weight on Western blotting to ~ 140-150 kDa. The specific ion and diuretic binding regions of the thiazide-sensitive Na/Cl cotransporter have not been distinguished.

Tubular (Jrine

Tubular (Jrine

Thiazide Sensitive Nacl Cotransporter

FIGURE 12. Molecular model of the Na/Cl cotransporter expressed in apical membranes of the distal convoluted tubule. This protein is the target site for thiazide and thiazide-like diuretics. See Fig. 8 and Table 2 for further descriptions of the gene and protein.

outside

membrane 1

r" 7_T

uuv

^ inside

----' HOOC, ---~

FIGURE 12. Molecular model of the Na/Cl cotransporter expressed in apical membranes of the distal convoluted tubule. This protein is the target site for thiazide and thiazide-like diuretics. See Fig. 8 and Table 2 for further descriptions of the gene and protein.

Lessons from Mutations in the Distal Convoluted Tubule Na/Cl Cotransporter (Slcl2a3) Found in Human Disease

Lifton and coworkers at Yale University and the International Collaborative Study Group for Bartter-Like Syndromes as well as others have identified mutations in the Slcl2a3 (Na/Cl cotransporter) gene on human chromosome 16 in kindreds with the Gitelman variant of Bartter syndrome. It has been proposed that these mutations in the cotransporter gene result in Na/Cl cotransporter proteins that function poorly or not at all (i.e., these are loss-of-function mutations). Individuals with Gitelman's variant exhibit the same renal salt wasting with subsequent volume depletion and hypokalemic metabolic alkalosis as the other variants of Bartter's syndrome. Unlike the classic or antenatal variants, who are hypercalciuric, the Gitelman's variant individuals exhibit hypocalci-uria. The latter is characteristic of the Gitelman's variant and is used to distinguish this form from the classic Bartter's syndrome. The characteristic hypocal-ciuria of the Gitelman's variant can be understood from the model of NaCl reabsorption in the distal convoluted tubule shown in Fig. 11. Since the magnitudes of NaCl and Ca2+ reabsorption are inversely related to each other in the distal convoluted tubule, significant reductions in (or complete loss of) Na/Cl cotransporter function would result in enhanced Ca2+ reabsorption and con sequent hypocalciuria. Individuals with Gitehnan's variant also usually present with significant hypomagnesemia.

General Aspects

The group of drugs acting on the collecting duct, also called "potassium-sparing diuretics," constitute an important part of the modern diuretic armamentarium. Because of their potassium-sparing effect, these weak diuretics are often used in combination with loop or thiazide diuretics to reduce or avoid potassium loss and hypokalemia that can develop with the latter diuretics. The epithelial Na+ channel expressed in apical membranes of connecting tubule cells and principal cells of the collecting duct is the target of amiloride and triamterene (see Fig. 13).

Model of the Sodium Transport

The mechanism of Na+ reabsorption by the principal cell of the cortical collecting duct is shown in Fig. 14. Sodium enters the cell across the apical plasma membrane down its electrochemical gradient through highly Na+ selective pores or channels. The favorable electrochemical gradient for Na+ entry is

DIURETICS AFFECTING COLLECTING DUCT SALT TRANSPORT

o amiloride (Midamor)

triamterene (Dyrenium)

FIGURE 13. Chemical structures of diuretics inhibiting Na+ transport in the cortical collecting duct (Site 4).

achieved by active extrusion of Na+ from the cell across the basolateral membrane via Na+/K+/ATPase.

Other Physiological Consequences of Inhibiting NaCl Absorption in the Collecting Duct

The major physiological effect of inhibiting Na+ entry into principal cells is the associated reduction in potassium secretion. Inhibition of Na+ channel activity results in depolarization of the voltage across the apical cell membrane (and also reduction in the lumen-negative transepithelial voltage) and a reduction in K+ entry into principal cells via the basolateral Na+/K+ATPase. These effects result in a diminished driving force for K+ secretion. The decrease in Na+/K+/ ATPase activity is a consequence of a lower intracellular Na+ activity resulting from diminished Na+ entry.

Modulation of active K+ secretion can also occur when nonreabsorbable charged compounds are present in the collecting duct tubular urine. Sulfate, phosphate, and anionic antibiotics (e.g., many penicillin derivatives) enhance the secretion of K+ by increasing lumen electrical negativity. Conversely, trimethoprim, an organic cationic antibiotic, reduces K+ secretion. Trimethoprim is often used for infection prophylaxis of AIDS patients and a common side-effect of this therapy is hyperkalemia. Recent studies demonstrate that trimethoprim mimics amiloride and triamterene and blocks apical membrane Na+

channels in the mammalian distal nephron and collecting duct. As a consequence, the transepithelial lumen-negative voltage is reduced and potassium secretion is inhibited. In addition, trimethoprim-mediated inhibition of baso-lateral Na+/K+/ATPase activity may also contribute to the reduced K+ secretion in collecting ducts.

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