Search

The degree to which loss of the NHE3 [Na.sup.+]/[H.sup.+] exchanger in the kidney contributes to impaired [Na.sup.+]-fluid volume homeostasis in NHE3-deficient ([Nhe3.sup.-/-]) 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.sup.-/-] mice. Transgenic [Nhe3.sup.-/-] ([tgNhe3.sup.-/-]) mice tolerated dietary NaCl depletion better than nontransgenic knockouts and showed no evidence of renal salt wasting. Unlike nontransgenic [Nhe3.sup.-/-] mice, [tgNhe3.sup.-/-] mice tolerated a 5% NaCl diet. When fed a 5% NaCl diet, [tgNhe3.sup.-/-] mice had lower serum aldosterone than [tgNhe3.sup.-/-] mice on a 1% NaCl diet, indicating improved extracellular fluid volume status. [Na.sup.+]-loaded [tgNhe3.sup.-/-] mice had sharply increased urinary [Na.sup.+] excretion, reflective of increased absorption of [Na.sup.+] 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.sup.-/-] 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.sup.+]-fluid volume homeostasis. sodium/hydrogen exchanger; diarrhea; Slc9a3; sodium absorption; sodium-fluid volume homeostasis

Sodium/proton exchangers [[Na.sup.+]/[H.sup.+] (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 NaC1 absorption, we measured radioisotopic [Na.sup.+] and [Cl.sup.-] 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.sup.+] (~6 [micro]eq/[cm.sup.2] * h) and [Cl.sup.-] (~3 [micro]eq/[cm.sup.2] * h) absorption. In contrast, NHE3(-) jejuna had reduced net [Na.sup.+] absorption (~2 [micro]eq/ cm.sup.2] * h) but absorbed [Cl.sup.-] at rates similar to NHE(+) and NHE2(-) jejuna. Treatment with 100 [micro]M 5-(N-ethyl-N-isopropyl) amiloride (EIPA) completely inhibited net [Na.sup.+] and [Cl.sup.-] absorption in all genotypes. Studies of the [Na.sup.+] absorptive flux [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] indicated that [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] in NHE(+) jejunum was not sensitive to 1 [micro]M EIPA, whereas [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] in NHE3(-)jejunum was equally sensitive to 1 and 100 [micro]M EIPA. Treatment with forskolin/ IBMX to increase intracellular cAMP ([cAMP.sub.i]) abolished net NaCl absorption and stimulated electrogenic [Cl.sup.-] 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.sup.+] absorption; 2) an amiloride-sensitive [Na.sup.+] transporter partially compensates for [Na.sup.+] absorption in NHE3(-) jejunum; 3) [cAMP.sub.i] stimulation abolishes net [Na.sup.+] absorption in NHE(+), NHE2(-), and NHE3(-)jejunum; and 4) electroneutral [Cl.sup.-] absorption is not directly dependent on either NHE2 or NHE3. sodium; chloride; cystic fibrosis transmembrane conductance regulator; adenosine 3',5'-cyclic monophosphate; chloridebicarbonate exchanger; sodium-hydrogen exchange; jejunum; small intestine; cystic fibrosis

The mechanisms involved in mediating the HCO3- reabsorption in the proximal tubules of NHE3 null mice were investigated. Results indicated that neither NHE2 nor any other ethylisopropylamiloride-sensitive NHE isoform contributes to mediating HCO3- reabsorption in the proximal tubule in the absence of NHE3. However, a bafilomycin-sensitive H+-ATPase was found to mediate a significant component of HCO3- reabsorption.

Mice lacking the NHE-3 gene show significant reduction in bicarbonate reabsorption in the proximal tubule. However, there was an increase in the net bicarbonate flux in cortical collecting duct (CCD) and the outer medullary collecting duct (OMCD) of the NHE-3 knockout mice. The H+-ATPase activity was responsible for increasing the net flux in the CCD while HKAg and the H+-ATPase contributed to the rise in the net flux in the OMCD.

To examine the function of Na+/H+ exchanger isoform 1 (NHE1) during embryogenesis, the Nhe1 gene was targeted by replacing the sequence encoding transmembrane domains 6 and 7 with the neomycin resistance gene. Results show that NHE1 activity is dispensable for normal embryogenesis. However, postnatal growth of homozygous mutants is affected and begin to exhibit ataxia and seizures at 2 weeks of age and histological analysis reveals an abnormal morphology of the grandular gastric mucosa.

The effect of hydrogen-bonding (H-bonding) amino acids on the ouabain affinity of sodium- and potassium-activated adenosinetriphosphatase (Na,K-ATPase) was investigated. The study protocol involved site-directed mutagenesis to substitute the transmembrane residues with hydrogen-binding potential in the NA,K-ATPase alpha subunit with residues that cannot participate in H-bonding. The results showed that tyrosine-108 and cysteine-104 are major determinants of Na,K-ATPase affinity for ouabain.

To study the role of [Na.sup.+]/[H.sup.+] exchanger isoform 2 (NHE2) and isoform 3 (NHE3) in sodium-fluid volume homeostasis and renal [Na.sup.+] conservation, mice with Nhe2 ([Nhe2.sup.-/-]) and/or Nhe3 ([Nhe3.sup.-/-]) null mutations were fed a [Na.sup.+]-restricted diet, and urinary [Na.sup.+] excretion, blood pressure, systemic acid-base and electrolyte status, and renal function were analyzed. [Na.sup.+]-restricted [Nhe2.sup.-/-] mice, on either a wild-type or Nhe3 heterozygous mutant ([Nhe3.sup.+/-]) background, did not exhibit excess urinary [Na.sup.+] excretion. After 15 days of [Na.sup.+] restriction, blood pressure, fractional excretion of [Na.sup.+], and the glomerular filtration rate (GFR) of [Nhe2.sup.-/-][Nhe3.sup.+/-] mice were similar to those of [Nhe2.sup.+/+] and [Nhe3.sup.+/-] mice, and no metabolic disturbances were observed. [Nhe3.sup.-/-] mice maintained on a [Na.sup.+]-restricted diet for 3 days exhibited hyperkalemia, urinary salt wasting, acidosis, sharply reduced blood pressure and GFR, and evidence of hypovolemic shock. These results negate the hypothesis that NHE2 plays an important renal function in sodium-fluid volume homeostasis; however, they demonstrate that NHE3 is critical for systemic electrolyte, acid-base, and fluid volume homeostasis during dietary [Na.sup.+] restriction and that its absence leads to renal salt wasting. sodium absorption; sodium/hydrogen exchanger; slc9a2; slc9a3

Role of sodium/hydrogen exchanger isoform NHE3 in fluid secretion and absorption in mouse and rat cholangiocytes. Am J Physiol Gastrointest Liver Physiol 280: G247-G254, 2001.--[Na.sup.+]/[H.sup.+] exchanger (NHE) isoforms play important roles in intracellular pH regulation and in fluid absorption. The isoform NHE3 has been localized to apical surfaces of epithelia and in some tissues may facilitate the absorption of NaCl. To determine whether the apical isoform NHE3 is present in cholangiocytes and to examine whether it has a functional role in cholangiocyte fluid secretion and absorption, immunocytochemical studies were performed in rat liver with NHE3 antibodies and functional studies were obtained in isolated bile duct units from wild-type and NHE3 (-/-) mice after stimulation with forskolin, using video-microscopic techniques. Our results indicate that NHE3 protein is present on the apical membranes of rat cholangiocytes and on the canalicular membrane of hepatocytes. Western blots also detect NHE3 protein in rat cholangiocytes and isolated canalicular membranes. After stimulation with forskolin, duct units from NHE3 (-/-) mice fail to absorb the secreted fluid from the cholangiocyte lumen compared with control animals. Similar findings were observed in isolated bile duct units from wild-type mice and rats in the presence of the [Na.sup.+]/[H.sup.+] exchanger inhibitor 5-(N-ethyl-N-isopropyl)-amiloride. In contrast, we could not demonstrate absorption of fluid from the canalicular lumen of mouse or rat hepatocyte couplets after stimulation of secretion with forskolin. These findings indicate that NHE3 is located on the apical membrane of rat cholangiocytes and that this NHE isoform can function to absorb fluid from the lumens of isolated rat and mouse cholangiocyte preparations. bile secretion; bile duct epithelium; hepatocyte

Shull, Gary E., Marian L. Miller, and Patrick J. Schultheis. Lessons from Genetically Engineered Animal Models. VIII. Absorption and secretion of ions in the gastrointestinal tract. Am. J. Physiol. Gastrointest. Liver Physiol. 278: G185--G190, 2000.--Absorption and secretion of ions in gastrointestinal and other epithelial tissues require the concerted activities of ion pumps, channels, symporters, ante exchangers, which operate in coupled systems to mediate transepithelial transport. Our understanding of the identities, membrane locations, and biochemical activities of epithelial ion transporters has advanced significantly in recent years, but major gaps and uncertainties remain in our understanding of their physiological functions. Increasingly. this problem is being addressed by the analysis of mutant mouse models developed by gene targeting. In this review, we discuss gene knockout studies of the secretory isoform of the [Na.sup.+]-[K.sup.+]-[2Cl.sup.-] cotransporter, isoforms 1, 2, and 3 of the [Na.sup.+]/[H.sup.+] exchanger, and the colonic [H.sup.+]-[K.sup.+]-ATPase. This approach is leading to a clearer understanding of the functions of these transporters in the living animal. embryonic stem cells; acid secretion; chloride secretion; sodium absorption; potassium absorption

Lorenz, John N., Patrick J. Schultheis, Timothy Traynor, Gary E. Shull, and Jurgen Schnermann. Micropuncture analysis of single-nephron function in NHE3-deficient mice. Am. J. Physiol. 277 (Renal Physiol. 46): F447-F453, 1999.--The Na/H exchanger isoform 3 (NHE3) is expressed in the proximal tubule and thick ascending limb and contributes to the reabsorption of fluid and electrolytes in these segments. The contribution of NHE3 to fluid reabsorption was assessed by micropuncture in homozygous ([Nhe3.sup.-/-]) and heterozygous ([Nhe3.sub.+/-]) knockout mice, and in their wild-type (WT, [Nhe3.sup.+/+]) littermates. Arterial pressure was lower in the [Nhe3.sup.-/-] mice (89 [+ or -] 6 mmHg) compared with [Nhe3.sup.+/+] (118 [+ or -] 4) and [Nhe3.sup.+/-] (108 [+ or -] 5). Collections from proximal and distal tubules demonstrated that proximal fluid reabsorption was blunted in both [Nhe3.sup.+/-] and [Nhe3.sup.-/-] mice (WT, 4.2 [+ or -] 0.3; [Nhe3.sup.+/-], 3.4 [+ or -] 0.2; and [Nhe3.sup.-/-], 2.6 [+ or -] 0.3 nl/min; P [is less than] 0.05). However, distal delivery of fluid was not different among the three groups of mice (WT, 3.3 [+ or -] 0.4 nl/min; [Nhe3.sup.+/-], 3.3 [+ or -] 0.2 nl/min; and [Nhe3.sup.-/-], 3.0 [+ or -] 0.4 nl/min; P [is less than] 0.05). In [Nhe3.sup.-/-] mice, this compensation was largely attributable to decreased single-nephron glomerular filtration rate (SNGFR): 10.7 [+ or -] 0.9 nl/min in the [Nhe3.sup.+/+] vs. 6.6 [+ or -] 0.8 nl/min in the [Nhe3.sup.-/-], measured distally. Proximal-distal SNGFR differences in [Nhe3.sup.-/-] mice indicated that much of the decrease in SNGFR was due to activation of tubuloglomerular feedback (TGF), and measurements of stop-flow pressure confirmed that TGF is intact in [Nhe3.sup.-/-] animals. In contrast to [Nhe3.sup.-/-] mice, normalization of early distal flow rate in [Nhe3.sup.+/-] mice was not related to decreased SNGFR (9.9 [+ or -] 0.7 nl/min), but rather, to increased fluid reabsorption in the loop segment ([Nhe3.sup.+/+], 2.6 [+ or -] 0.2; [Nhe3.sup.+/-], 3.6 [+ or -] 0.5 nl/min). We conclude that NHE3 is a major Na/H exchanger isoform mediating [Na.sup.+] and fluid reabsorption in the proximal tubule. In animals with NHE3 deficiency, normalization of fluid delivery to the distal tubule is achieved through alterations in filtration rate and/or downstream transport processes. kidney; single-nephron glomerular filtration rate; sodium/ proton exchange; proximal tubule; tubuloglomerular feed-back