NHE3 is one of five plasma membrane Na+/H+ exchangers and is encoded by the mouse gene Slc9a3. It is expressed on apical membranes of renal proximal tubule and intestinal epithelial cells and is thought to play a major role in NaCl and HCO3- absorption. As the distribution of NHE3 overlaps with that of the NHE2 isoform in kidney and intestine, the function and relative importance of NHE3 in vivo is unclear. To analyse its physiological functions, we generated mice lacking NHE3 function. Homozygous mutant (Slc9a3-/-) mice survive, but they have slight diarrhoea and blood analysis revealed that they are mildly acidotic. HCO3- and fluid absorption are sharply reduced in proximal convoluted tubules, blood pressure is reduced and there is a severe absorptive defect in the intestine. Thus, compensatory mechanisms must limit gross perturbations of electrolyte and acid-base balance. Plasma aldosterone is increased in NHE3-deficient mice, and expression of both renin and the AE1 (Slc4a1) Cl-/HCO3- exchanger mRNAs are induced in kidney. In the colon, epithelial Na+ channel activity is increased and colonic H+,K+-ATPase mRNA is massively induced. These data show that NHE3 is the major absorptive Na+/H+ exchanger in kidney and intestine, and that lack of the exchanger impairs acid-base balance and Na+-fluid volume homeostasis.

In chloride-secretory epithelia, the basolateral Na-K2Cl cotransporter (NKCC1) is thought to play a major role in transepithelial Cl ؊ and fluid transport. Similarly, in marginal cells of the inner ear, NKCC1 has been proposed as a component of the entry pathway for K ؉ that is secreted into the endolymph, thus playing a critical role in hearing. To test these hypotheses, we generated and analyzed an NKCC1-deficient mouse. Homozygous mutant (Nkcc1 ؊/؊ ) mice exhibited growth retardation, a 28% incidence of death around the time of weaning, and mild difficulties in maintaining their balance. Mean arterial blood pressure was significantly reduced in both heterozygous and homozygous mutants, indicating an important function for NKCC1 in the maintenance of blood pressure. cAMP-induced short circuit currents, which are dependent on the CFTR Cl ؊ channel, were reduced in jejunum, cecum, and trachea of Nkcc1 ؊/؊ mice, indicating that NKCC1 contributes to cAMP-induced Cl ؊ secretion. In contrast, secretion of gastric acid in adult Nkcc1 ؊/؊ stomachs and enterotoxin-stimulated fluid secretion in the intestine of suckling Nkcc1 ؊/؊ mice were normal. Finally, homozygous mutants were deaf, and histological analysis of the inner ear revealed a collapse of the membranous labyrinth, consistent with a critical role for NKCC1 in transepithelial K ؉ movements involved in generation of the K ؉ -rich endolymph and the endocochlear potential.

The NBC1 Na ؉ /HCO 3 ؊ cotransporter is expressed in many tissues, including kidney and intestinal epithelia. NBC1 mutations cause proximal renal tubular acidosis in humans, consistent with its role in HCO 3 ؊ absorption in the kidney. In intestinal and colonic epithelia, NBC1 localizes to basolateral membranes and is thought to function in anion secretion. To test the hypothesis that NBC1 plays a role in transepithelial HCO 3 ؊ secretion in the intestinal tract, null mutant (NBC1 /HCO 3 Ϫ cotransporters (1-3). NBC1 has two protein variants, which localize to basolateral membranes (4) and mediate electrogenic Na ϩ /HCO 3 Ϫ cotransport (2, 3). The kNBC1 variant is expressed in kidney epithelia and eye (4, 5), and the pNBC1 variant is expressed in pancreas, duodenum, colon, and several other tissues (4 -8). The stoichiometry of the transporter can be altered from 1NaϪ by phosphorylation of a residue near the carboxyl terminus (9). In the kidney, the ion stoichiometry and electrochemical driving forces for NBC1 result in Na ϩ and HCO 3 Ϫ extrusion across the basolateral membrane (2, 3, 9, 10); thus, in the kidney, NBC1 functions in HCO 3 Ϫ reabsorption in the proximal tubule (2, 3). In pancreas and the intestinal tract, the ion stoichiometry and driving forces for NBC1 appear to result in Na ϩ and HCO 3 Ϫ entry into the cell (2, 8); thus, in intestine and colon, NBC1 has been proposed to mediate HCO 3 Ϫ uptake across the basolateral membrane to support transepithelial anion secretion (8, 11).Human patients with proximal renal tubular acidosis resulting from mutations in NBC1 have been reported (12-16), thereby confirming a bicarbonate-absorptive role for NBC1 in kidney. The primary mutations were single amino acid substitutions (R298S, T485S, R510H, A799V, R881C, and S427L), which appeared to cause decreased function of the cotransporter rather than loss of function (12)(13)(14). One patient had an inactivating mutation in the unique N terminus of the kidney NBC1 variant (Q29X), but the pancreatic variant, which is expressed in many other tissues and at low levels in kidney (4), was intact (15). Only a single patient has been identified with a complete inactivating mutation, a nucleotide deletion that causes a frameshift at codon 721 (16). The pRTA resulting from NBC1 mutations clearly shows that this transporter is essential for renal HCO 3 Ϫ absorption; however, clinically significant intestinal disease has not been reported.NBC1 has been localized to the basolateral membrane of epithelial cells lining both the small and large intestine (8,17,18). In the colon, its expression was greatest in crypt cells, consist-* This work was supported by National Institutes of Health (NIH) Grants DK50594 and HL61974 (to G. E. S.), DK67749 (to L. R. G.), DK57552 (to J. N. L.), DK48816 (to L. L. C.), and T32-RR-07004 (to J. E. S.) and NIEHS, NIH, Grant ES06096 (to the Center for Environmental Genetics, Alvaro Puga PI). The costs of publication of this article were defrayed in part by the payment of page charges. This article mus...

The AE2 Cl ؊ /HCO 3 ؊ exchanger is expressed in numerous cell types, including epithelial cells of the kidney, respiratory tract, and alimentary tract. In gastric epithelia, AE2 is particularly abundant in parietal cells, where it may be the predominant mechanism for HCO 3 ؊ efflux and Cl ؊ influx across the basolateral membrane that is needed for acid secretion. To investigate the hypothesis that AE2 is critical for parietal cell function and to assess its importance in other tissues, homozygous null mutant (AE2 ؊/؊ ) mice were prepared by targeted disruption of the AE2 (Slc4a2) gene. AE2 ؊/؊ mice were emaciated, edentulous (toothless), and exhibited severe growth retardation, and most of them died around the time of weaning. AE2 ؊/؊ mice exhibited achlorhydria, and histological studies revealed abnormalities of the gastric epithelium, including moderate dilation of the gastric gland lumens and a reduction in the number of parietal cells. There was little evidence, however, that parietal cell viability was impaired. Ultrastructural analysis of AE2 ؊/؊ gastric mucosa revealed abnormal parietal cell structure, with severely impaired development of secretory canaliculi and few tubulovesicles but normal apical microvilli. These results demonstrate that AE2 is essential for gastric acid secretion and for normal development of secretory canalicular and tubulovesicular membranes in mouse parietal cells.

Sodium/proton exchangers [Na(+)/H(+) (NHEs)] play an important role in salt and water absorption from the intestinal tract. To investigate the contribution of the apical membrane NHEs, NHE2 and NHE3, to electroneutral NaCl absorption, we measured radioisotopic Na(+) and Cl(-) flux across isolated jejuna from wild-type [NHE(+)], NHE2 knockout [NHE2(-)], and NHE3 knockout [NHE3(-)] mice. Under basal conditions, NHE(+) and NHE2(-) jejuna had similar rates of net Na(+) (approximately 6 microeq/cm(2) x h) and Cl(-) (approximately 3 microeq/cm(2) x h) absorption. In contrast, NHE3(-) jejuna had reduced net Na(+) absorption (approximately 2 microeq/cm(2) x h) but absorbed Cl(-) at rates similar to NHE(+) and NHE2(-) jejuna. Treatment with 100 microM 5-(N-ethyl-N-isopropyl) amiloride (EIPA) completely inhibited net Na(+) and Cl(-) absorption in all genotypes. Studies of the Na(+) absorptive flux (J) indicated that J in NHE(+) jejunum was not sensitive to 1 microM EIPA, whereas J in NHE3(-) jejunum was equally sensitive to 1 and 100 microM EIPA. Treatment with forskolin/IBMX to increase intracellular cAMP (cAMP(i)) abolished net NaCl absorption and stimulated electrogenic Cl(-) secretion in all three genotypes. Quantitative RT-PCR of epithelia from NHE2(-) and NHE3(-) jejuna did not reveal differences in mRNA expression of NHE3 and NHE2, respectively, when compared with jejunal epithelia from NHE(+) siblings. We conclude that 1) NHE3 is the dominant NHE involved in small intestinal Na(+) absorption; 2) an amiloride-sensitive Na(+) transporter partially compensates for Na(+) absorption in NHE3(-) jejunum; 3) cAMP(i) stimulation abolishes net Na(+) absorption in NHE(+), NHE2(-), and NHE3(-) jejunum; and 4) electroneutral Cl(-) absorption is not directly dependent on either NHE2 or NHE3.