Na+:HCO(3-) cotransporters (NBC): cloning and characterization.

The sodium bicarbonate cotransporter (NBC1) is essential for bicarbonate transport across plasma membranes in epithelial and nonepithelial cells. The direction of the NaHCO3 movement in secretory epithelia is opposite to that in reabsorptive epithelia. In secretory epithelia (such as pancreatic duct cells) NBC is responsible for the transport of bicarbonate from blood to the cell for eventual secretion at the apical membrane. In reabsorptive epithelia (such as kidney proximal tubule cells) NBC is responsible for the reabsorption of bicarbonate from cell to the blood. In nonepithelial cells this transporter is mainly involved with cell pH regulation. Recent molecular cloning experiments have identified the existence of four NBC isoforms (NBC1, 2, 3 and 4) and two NBC-related proteins AE4 and NCBE (Anion Exchanger 4 and Na-dependent Chloride-Bicarbonate Exchanger). All but AE4 are presumed to mediate the cotransport of Na+ and HCO(3-) under normal conditions and may be functionally altered in certain pathologic states. NBC1 shows a limited tissue expression pattern, is electrogenic and plays an important role in bicarbonate reabsorption in kidney proximal tubule. In addition to the kidney, NBC1 is expressed in pancreatic duct cells, is activated by cystic fibrosis transmembrane conductance regulator (CFTR) and plays an important role in HCO3- secretion. NBC2 and NBC3 have a wider tissue distribution than NBC1, are electroneutral, and are involved with cell pH regulation. The characterization of NBC4 is incomplete. The NBC-related protein called NCBE mediates Na-dependent, Cl-/Bicarbonate Exchange. The purpose of this review is to summarize recent advances on the cloning of NBC isoforms and related proteins and their role and regulation in physiologic and pathologic states.

Mouse Na+: HCO3- cotransporter isoform NBC-3 (kNBC-3): cloning, expression, and renal distribution.

BACKGROUND: Na+:HCO3- cotransporters mediate the transport of HCO3- into or out of the cell. We recently reported the partial cloning and characterization of a new human Na+:HCO3- cotransporter (referred to as NBC-3 or kNBC-3). The purpose of the present studies was to clone the mouse kNBC-3 and to examine its properties and expression in the kidney. METHODS: Using primers from human kNBC-3 cDNA and 5' and 3' rapid amplification cDNA end polymerase chain reaction (RACE PCR), the mouse kNBC-3 full-length cDNA was cloned from inner medullary collecting duct (mIMCD-3) cells. The tissue distribution and functional properties of NBC-3 was determined using established methods. RESULTS: The coding region of the mouse kNBC-3 has 1089 amino acids and shows 73 and 56% identity to human NBC-2 and NBC-1, respectively. The renal distribution of kNBC-3 demonstrated a unique expression pattern: Whereas kNBC-1 is predominantly expressed in the cortex and is absent in the inner medulla, kNBC-3 shows an intense expression level in the inner medulla and is absent in the cortex. Expression studies in oocytes indicated that NBC-3 mediates Na-dependent HCO3- cotransport. Electrophysiological experiments demonstrated that unlike kNBC-1, which is electrogenic, kNBC-3 is electroneutral. CONCLUSIONS: Based on its distribution and electroneutrality, we propose that kNBC-3 mediates the transport of HCO3- into the cells.

Pendrin: an apical Cl-/OH-/HCO3- exchanger in the kidney cortex.

The identities of the apical Cl-/base exchangers in kidney proximal tubule and cortical collecting duct (CCD) cells remain unknown. Pendrin (PDS), which is expressed at high levels in the thyroid and its mutation causes Pendred's syndrome, is shown to be an anion exchanger. We investigated the renal distribution of PDS and its function. Our results demonstrate that pendrin mRNA expression in the rat kidney is abundant and limited to the cortex. Proximal tubule suspensions isolated from kidney cortex were highly enriched in pendrin mRNA. Immunoblot analysis studies localized pendrin to cortical brush-border membranes. Nephron segment RT-PCR localized pendrin mRNA to proximal tubule and CCD. Expression studies in HEK-293 cells demonstrated that pendrin functions in the Cl-/OH-, Cl-/HCO3-, and Cl-/formate exchange modes. The conclusion is that pendrin is an apical Cl-/base exchanger in the kidney proximal tubule and CCD and mediates Cl-/OH-, Cl-/HCO3-, and Cl-/formate exchange.

Glucocorticoids enhance the expression of the basolateral Na+:HCO3- cotransporter in renal proximal tubules.

BACKGROUND: Studies have shown that glucocorticoids enhance HCO3- reabsorption in proximal tubules. Functional and molecular studies indicate that HCO3- reabsorption in proximal tubules is mediated via luminal H(+)-ATPase and Na+/H+ exchanger (NHE-3), and basolateral Na+:HCO3- cotransporter (NBC) acting in series. The purpose of these experiments was to examine the effect of adrenal steroids on NBC-1 and NHE-3 expression and activity in rat renal proximal tubules. METHODS: Rats were injected subcutaneously with dexamethasone (100 mu/day) or deoxycorticosterone acetate (30 mg/kg), potent glucocorticoid, or mineralocorticoid analogues, respectively. Animals were sacrificed after two or four days, and NBC-1 and NHE-3 mRNA expression and activities were measured in cortex and proximal tubules. RESULTS: Northern hybridizations indicated that cortical NBC-1 mRNA expression increased by approximately 92% in rats treated with dexamethasone for four days (N = 6, P < 0.03) but not two days. NHE-3 mRNA expression remained unchanged. NBC and NHE-3 activities were measured as the Na-dependent pHi recovery from an acid load in the presence or absence of HCO3-, respectively, and appropriate inhibitors in proximal tubule suspensions loaded with BCECF. NBC activity increased by approximately 80% in rats treated with dexamethasone for four days (P < 0.01, N = 5) but not two days. NHE-3 activity increased by 34 and 42% in rats treated with dexamethasone for two and four days, respectively (P < 0.05 and P < 0.02 for each group vs. control). Treatment with deoxycorticosterone acetate did not alter NBC-1 expression. CONCLUSION: Glucocorticoids at pharmacologic concentrations enhance the mRNA expression and functional activity of renal proximal tubule NBC-1. Enhanced NBC and NHE-3 activities could result in increased HCO3- reabsorption in proximal tubule and could contribute to the maintenance of metabolic alkalosis in pathophysiologic states associated with increased glucocorticoid production.

Physiologic and molecular aspects of the Na+:HCO3- cotransporter in health and disease processes.

Approximately 80% of the filtered load of HCO3- is reabsorbed in the proximal tubule via a process of active acid secretion by the luminal membrane. The major mechanism for the transport of HCO3- across the basolateral membrane is via the electrogenic Na+:3HCO3- cotransporter (NBC). Recent molecular cloning experiments have identified the existence of three NBC isoforms (NBC-1, NBC-2, and NBC-3).1 Functional and molecular studies indicate the presence of all three NBC isoforms in the kidney. All are presumed to mediate the cotransport of Na+ and HCO3- under normal conditions and may be functionally altered in certain pathophysiologic states. Specifically, NBC-1 may be up-regulated in metabolic acidosis and potassium depletion and in response to glucocorticoid excess and may be down-regulated in response to HCO3- loading or alkalosis. Recent studies provide molecular evidence indicating the expression of NBC-1 in pancreatic duct cells. NBC is activated by cystic fibrosis transmembrane conductance regulator (CFTR) and plays an important role in HCO3- secretion in the agonist-stimulated state in pancreatic duct cells. The purpose of this review is to summarize recent functional and molecular studies on the regulation of NBCs in physiologic and pathophysiologic states. Possible signals responsible for the regulation of NBCs in these conditions are examined. Furthermore, the possible role of this transporter in acid-base disorders (such as proximal renal tubular acidosis) is discussed.

Characterization of Na+/HCO-3 cotransporter isoform NBC-3.

Na+-HCO-3 cotransporters mediate the transport of HCO-3 into or out of the cell. Two Na+-HCO-3 cotransporters (NBC) have been identified previously, which are referred to as NBC-1 and NBC-2. A cDNA library from uninduced human NT-2 cells was screened with an NBC-2 cDNA probe. Several clones were identified and isolated. Sequence analysis of these clones identified a partial coding region (2 kb) of a novel NBC (called here NBC-3), which showed 53% and 72% identity with NBC-1 and NBC-2, respectively. Northern blot analysis revealed that NBC-3 encodes a 4.4-kb mRNA with a tissue distribution pattern distinct from NBC-1 and NBC-2. NBC-3 is highly expressed in brain and spinal column, with moderate levels in trachea, thyroid, and kidney. In contrast with NBC-1, NBC-3 shows low levels of expression in pancreas and kidney cortex. In the kidney, NBC-3 expression is predominantly limited to the medulla. Cultured mouse inner medullary collecting duct (mIMCD-3) cells showed high levels of NBC-1 and low levels of NBC-3 mRNA expression. Subjecting the mutagenized mIMCD-3 cells to sublethal acid stress decreased the mRNA expression of NBC-1 by approximately 90% but increased the Na+-dependent HCO-3 cotransport activity by approximately 7-fold (as assayed by DIDS-sensitive, Na+-dependent, HCO-3-mediated intracellular pH recovery). This increase was associated with approximately 5.5-fold enhancement of NBC-3 mRNA levels. NBC showed significant affinity for Li+ in the mutant but not the parent mIMCD-3 cells. On the basis of the widespread distribution of NBC-3, we propose that this isoform is likely involved in cell pH regulation by transporting HCO-3 from blood to the cell. We further propose that enhanced expression of NBC-3 in severe acid stress could play an important role in cell survival by mediating the influx of HCO-3 into the cells.

Cloning, renal distribution, and regulation of the rat Na+-HCO3- cotransporter.

We recently reported the cloning and expression of a human kidney Na+-HCO3- cotransporter (NBC-1) (C. E. Burnham, H. Amlal, Z. Wang, G. E. Shull, and M. Soleimani. J. Biol. Chem. 272: 19111-19114, 1997). To expedite in vivo experimentation, we now report the cDNA sequence of rat kidney NBC-1. In addition, we describe both the organ and nephron segment distributions and the regulation of NBC-1 mRNA under three models of pH stress: chloride-depletion alkalosis (CDA), metabolic acidosis, and bicarbonate loading. Rat NBC-1 cDNA encodes an open reading frame of 1,035 amino acids, with 96 and 87% identity to human and salamander NBC-1, respectively. Rat NBC-1 mRNA is expressed at high levels in kidney and brain, with lower levels in colon, stomach, and heart. None appears in liver. In the kidney, NBC-1 is expressed mainly in the proximal tubule, with traces found in medullary thick ascending limb and papilla. HCO3- loading decreased NBC-1 mRNA levels, which were unchanged either by metabolic acidosis or by CDA.

Cloning and functional expression of a human kidney Na+:HCO3- cotransporter.

Several modes of HCO3- transport occur in the kidney, including Na+-independent Cl/HCO3- exchange (mediated by the AE family of Cl-/HCO3- exchangers), sodium-dependent Cl-/HCO3- exchange, and Na+:HCO3- cotransport. The functional similarities between the Na+-coupled HCO3- transporters and the AE isoforms (i.e. transport of HCO3- and sensitivity to inhibition by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid) suggested a strategy for cloning the other transporters based on structural similarity with the AE family. An expressed sequence tag encoding part of a protein that is related to the known anion exchangers was identified in the GenBankTM expressed sequence tag data base and used to design an oligonucleotide probe. This probe was used to screen a human kidney cDNA library. Several clones were identified, isolated, and sequenced. Two overlapping cDNA clones were spliced together to form a 7.6-kilobase cDNA that contained the entire coding region of a novel protein. Based on the deduced amino acid sequence, the cDNA encodes a protein with a Mr of 116,040. The protein has 29% identity with human brain AE3. Northern blot analysis reveals that the 7.6-kilobase mRNA is highly expressed in kidney and pancreas, with detectable levels in brain. Functional studies in transiently transfected HEK-293 cells demonstrate that the cloned transporter mediates Na+:HCO3- cotransport.

A liver-specific isoform of the betaine/GABA transporter in the rat: cDNA sequence and organ distribution.

We report the cloning of a 2.2 kb cDNA encoding a Na(+)-and Cl- dependent betaine/GABA (gamma-aminobutyric acid) transporter from rat liver poly(A+) RNA. 5'-RACE revealed an additional 355 bases 5' to the 2.2 kb cDNA sequence. Northern analysis demonstrated two (2.2 kb and 2.6 kb) mRNA isoforms in rat liver. Betaine transporter mRNA was also detected in the brain, spleen, lung, and kidney using the 2.2 kb cDNA clone as a probe. Only the 2.6 kb mRNA from the liver hybridized with the 5'-RACE product.

Expression of the Na(+)-K(+)-2Cl- cotransporter in Xenopus oocytes.

The Na(+)-K(+)-2Cl- cotransporter (NKCCT) is important in mediating net salt transport in a variety of cells. As a first step in obtaining structural information on this transport system, we attempted to express it in Xenopus laevis oocytes by injection of poly(A)+ mRNA from rat kidney. Four days after injection, batches of oocytes were tested for 86Rb uptake in presence and absence of 10 microM bumetanide to distinguish that fraction of influx mediated by NKCCT. In the absence of bumetanide, the oocytes formed a bimodal distribution with respect to 86Rb uptake, with some oocytes accumulating significantly more 86Rb than others. In the presence of bumetanide, or when Na was replaced with choline, that group of oocytes taking up more 86Rb did not appear. Sham-injected oocytes did not accumulate sufficient 86Rb to be distinguishable above background. Taken together, these data suggest that some of the mRNA-injected oocytes expressed the rat renal NKCCT. Crude poly(A)+ mRNA was separated by acid-urea agarose gel electrophoresis, and fractions were injected into oocytes. One fraction corresponding to messages of approximately 7 kilobases in length induced a bumetanide-sensitive 86Rb influx resembling that seen with total mRNA. Poly(A)+ mRNA fractionated on sucrose density gradients gave similar results. It is concluded that the rat kidney NKCCT has been expressed in Xenopus oocytes from a high molecular weight fraction of poly(A)+ mRNA.

Molecular cloning of rat renin cDNA and its gene.

Renin (EC 3.4.23.15) is an aspartyl protease that cleaves its only known substrate, angiotensinogen, to release the vasopressor hormone angiotensin. We have isolated full-length cDNAs for renin from a rat kidney cDNA library. The cDNAs are complementary to a 1434-nucleotide rat kidney mRNA that encodes preprorenin, the 402-amino acid precursor of renin. This rat cDNA was used to isolate the complete copy of a renin gene from a rat genomic library, and a comparison of this genomic clone with rat genomic DNA showed that renin is a single-copy gene in the rat. Rat renin is 85% identical to one mouse renin isozyme (renin-1) and 81% identical to the second mouse renin isozyme (renin-2), suggesting that the duplication of the mouse renin genes is a more recent event than the speciation of rats and mice. Analyses of rat, human, and mouse renin sequences revealed that the potential to form two-chain renin is apparently peculiar to mouse renin, and the expression of a tenth exon (which results in a three-amino acid insertion) is observed only in the human renin gene.

Edentulous persons, United States-1971.

There were an estimated 22.6 million edentulous persons in the United States according to the Health Interview Survey conducted in 1971. The Ju1y 1957-June 1958 survey estimate of the number of persons who were edentulous (had lost all their natural teeth) was 21.9 million. Since in most instances it takes many years of dental neglect for an individual to lose all his teeth, persons with no teeth are heavily concentrated in the older age groups. For example, in 1971,86.3 percent (19.5 million) of the edentulous population were in the age group 45 years of age and older, while in 1957-58 the comparable figure was 85.2 percent. Virtually every American will be affected during his or her lifetime by dental decay or periodontal disease. Reports previously published in the Vital and Health Statistics series contain national estimates of the prevalence, severity, and effects of dental disease among U.S. adults. Based on dental examinations conducted during 1960-62, it was estimated that approximately 20 million adults had lost all their natural teeth. Of the remaining adults with at least one natural tooth (approximately 90 million), about haIf had 18 or more decayed, missing, or filled teeth. In addition, about three out of four of those with natural teeth had periodontal disease, and about one out of four had an advanced form of periodontal disease with pocket formations. The result of neglected dental caries and advanced periodontal disease is the loss of teeth. Persons using artificial dentures are considerably less efficient at chewing than persons with healthy natural teeth. For the aged, artificial dentures mean a decrease in masticator function at a time when an efficient dental function is increasingly desirable due to changes in nutritional requirements. While the ultimate dental health goaI is the eradication of dental disease, an important immediate goal is the reduction of tooth Ioss. Tooth loss can usualIy be obviated by diagnosing and treating dental disease in its early stages and by utilizing the preventive measures now available.