Colonic anion secretory defects and metabolic acidosis in mice lacking the NBC1 Na+/HCO3- cotransporter.

The NBC1 Na+/HCO3- 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 HCO3- 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 HCO3- secretion in the intestinal tract, null mutant (NBC1-/-) mice were prepared by targeted disruption of its gene (Slc4a4). NBC1-/- mice exhibited severe metabolic acidosis, growth retardation, reduced plasma Na+, hyperal-dosteronism, splenomegaly, abnormal dentition, intestinal obstructions, and death before weaning. Intracellular pH (pH(i)) was not altered in cAMP-stimulated epithelial cells of NBC1-/- cecum, but pH(i) regulation during sodium removal and readdition was impaired. Bioelectric measurements of NBC1-/- colons revealed increased amiloride-sensitive Na+ absorption. In Ringer solution containing both Cl- and HCO3-, the magnitude of cAMP-stimulated anion secretion was normal in NBC1-/- distal colon but increased in proximal colon, with the increase largely supported by enhanced activity of the basolateral NKCC1 Na+-K+-2Cl- cotransporter. Anion substitution studies in which carbonic anhydrase was inhibited and transepithelial anion conductance was limited to HCO3- revealed a sharp decrease in both cAMP-stimulated HCO3- secretion and SITS-sensitive current in NBC1-/- proximal colon. These results are consistent with the known function of NBC1 in HCO3- absorption in the kidney and demonstrate that NBC1 activity is a component of the basolateral mechanisms for HCO3- uptake during cAMP-stimulated anion secretion in the proximal colon.

Blood pressure maintenance in NHE3-deficient mice with transgenic expression of NHE3 in small intestine.

NHE3 Na(+)/H(+) exchanger knockout (Nhe3(-/-)) mice have severe absorptive deficits in the kidney proximal tubule and intestinal tract. The resulting hypovolemia has confounded efforts to carefully evaluate the specific effects of NHE3 deficiency on kidney function. Development of mice with transgenic expression of NHE3 in the small intestine (tgNhe3(-/-)) has allowed us to analyze the role of renal NHE3 in overall maintenance of blood pressure, pressure natriuresis, and autoregulation of both glomerular filtration rate (GFR) and renal blood flow (RBF). Ambulatory blood pressure, measured by telemetry, was lower in tgNhe3(-/-) mice than in wild-type controls (tgNhe3(+/+)) when the mice were maintained on a normal NaCl diet but was normalized when they were provided with a high NaCl intake. Furthermore, administration of the AT1-receptor blocker losartan showed that circulating ANG II plays a major role in maintaining blood pressure in tgNhe3(-/-) mice fed normal NaCl but not in those receiving high NaCl. Clearance studies revealed a blunted pressure-natriuresis response in tgNhe3(-/-) mice at lower blood pressures but a robust response at higher blood pressures. Autoregulation of GFR and RBF was normal in tgNhe3(-/-) mice. These results show that dietary NaCl loading normalizes blood pressure in awake tgNhe3(-/-) mice and that alterations in NHE3 activity are not essential for normal autoregulation of GFR and RBF. Furthermore, the data strongly support the hypothesis that NHE3 plays an important role in the diuretic and natriuretic responses to increases in blood pressure but also show that mechanisms not involving NHE3 mediate pressure natriuresis in the higher range of blood pressures studied.

Roles of the Na,K-ATPase alpha4 isoform and the Na+/H+ exchanger in sperm motility.

The Na,K-ATPase generates electrochemical gradients that are used to drive the coupled transport of many ions and nutrients across the plasma membrane. The functional enzyme is comprised of an alpha and beta subunit and families of isoforms for both subunits exist. Recent studies in this laboratory have identified a biological role for the Na,K-ATPase alpha4 isoform in sperm motility. Here we further investigate the role of the Na,K-ATPase carrying the alpha4 isoform, showing again that ouabain eliminates sperm motility, and in addition, that nigericin, a H+/K+ ionophore, and monensin, a H+/Na+ ionophore, reinitiate motility. These data, along with the observation that the K+ ionophore valinomycin has no effect on the motility of ouabain-inhibited sperm, suggest that ouabain may change intracellular H+ levels in a manner that is incompatible with sperm motility. We have also localized NHE1 and NHE5, known regulators of intracellular H+ content, to the same region of the sperm as the Na,K-ATPase alpha4 isoform. These data highlight the important role of the Na,K-ATPase alpha4 isoform in regulating intracellular H(+) levels, and provide evidence suggesting the involvement of the Na+/H+ exchanger, which is critical for maintaining normal sperm motility.

Intestinal NaCl transport in NHE2 and NHE3 knockout mice.

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.

Renal function in NHE3-deficient mice with transgenic rescue of small intestinal absorptive defect.

The degree to which loss of the NHE3 Na(+)/H(+) exchanger in the kidney contributes to impaired Na(+)-fluid volume homeostasis in NHE3-deficient (Nhe3(-/-)) mice is unclear because of the coexisting intestinal absorptive defect. To more accurately assess the renal effects of NHE3 ablation, we developed a mouse with transgenic expression of rat NHE3 in the intestine and crossed it with Nhe3(-/-) mice. Transgenic Nhe3(-/-) (tgNhe3(-/-)) mice tolerated dietary NaCl depletion better than nontransgenic knockouts and showed no evidence of renal salt wasting. Unlike nontransgenic Nhe3(-/-) mice, tgNhe3(-/-) mice tolerated a 5% NaCl diet. When fed a 5% NaCl diet, tgNhe3(-/-) mice had lower serum aldosterone than tgNhe3(-/-) mice on a 1% NaCl diet, indicating improved extracellular fluid volume status. Na(+)-loaded tgNhe3(-/-) mice had sharply increased urinary Na(+) excretion, reflective of increased absorption of Na(+) in the small intestine; nevertheless, they remained hypotensive, and renal studies showed a reduction in glomerular filtration rate (GFR) similar to that observed in nontransgenic Nhe3(-/-) mice. These data show that reduced GFR, rather than being secondary to systemic hypovolemia, is a major renal compensatory mechanism for the loss of NHE3 and indicate that loss of NHE3 in the kidney alters the set point for Na(+)-fluid volume homeostasis.

Mice with a targeted disruption of the AE2 Cl-/HCO3- exchanger are achlorhydric.

The AE2 Cl-/HCO3- 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 HCO3- 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.

In vivo evidence for interferon-gamma-mediated homeostatic mechanisms in small intestine of the NHE3 Na+/H+ exchanger knockout model of congenital diarrhea.

Mice lacking NHE3, the major absorptive Na(+)/H(+) exchanger in the intestine, are the only animal model of congenital diarrhea. To identify molecular changes underlying compensatory mechanisms activated in chronic diarrheas, cDNA microarrays and Northern blot analyses were used to compare global mRNA expression patterns in small intestine of NHE3-deficient and wild-type mice. Among the genes identified were members of the RegIII family of growth factors, which may contribute to the increased absorptive area, and a large number of interferon-gamma-responsive genes. The latter finding is of particular interest, since interferon-gamma has been shown to regulate ion transporter activities in intestinal epithelial cells. Serum interferon-gamma was elevated 5-fold in NHE3-deficient mice; however, there was no evidence of inflammation, and unlike conditions such as inflammatory bowel disease, levels of other cytokines were unchanged. In addition, quantitative PCR analysis showed that up-regulation of interferon-gamma mRNA was localized to the small intestine and did not occur in the colon, spleen, or kidney. These in vivo data suggest that elevated interferon-gamma, produced by gut-associated lymphoid tissue in the small intestine, is part of a homeostatic mechanism that is activated in response to the intestinal absorptive defect in order to regulate the fluidity of the intestinal tract.