Search

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 withNhe3−/−mice. TransgenicNhe3−/−(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.

To study the role of Na+/H+exchanger isoform 2 (NHE2) and isoform 3 (NHE3) in sodium-fluid volume homeostasis and renal Na+conservation, mice with Nhe2(Nhe2−/−) and/or Nhe3(Nhe3−/−) null mutations were fed a Na+-restricted diet, and urinary Na+excretion, blood pressure, systemic acid-base and electrolyte status, and renal function were analyzed. Na+-restrictedNhe2−/−mice, on either a wild-type orNhe3heterozygous mutant (Nhe3+/−) background, did not exhibit excess urinary Na+excretion. After 15 days of Na+restriction, blood pressure, fractional excretion of Na+, and the glomerular filtration rate (GFR) ofNhe2−/−Nhe3+/−mice were similar to those of Nhe2+/+andNhe3+/−mice, and no metabolic disturbances were observed. Nhe3−/−mice maintained on a Na+-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+restriction and that its absence leads to renal salt wasting.

Multiple Na+/H+exchangers (NHEs) are expressed in salivary gland cells; however, their functions in the secretion of saliva by acinar cells and the subsequent modification of the ionic composition of this fluid by the ducts are unclear. Mice with targeted disruptions of the Nhe1,Nhe2, and Nhe3genes were used to study the in vivofunctions of these exchangers in parotid glands. Immunohistochemistry indicated that NHE1 was localized to the basolateral and NHE2 to apical membranes of both acinar and duct cells, whereas NHE3 was restricted to the apical region of duct cells. Na+/H+exchange was reduced more than 95% in acinar cells and greater than 80% in duct cells of NHE1-deficient mice (Nhe1−/−). Salivation in response to pilocarpine stimulation was reduced significantly in bothNhe1−/−and Nhe2−/−mice, particularly during prolonged stimulation, whereas the loss of NHE3 had no effect on secretion. Expression of Na+/K+/2Cl−cotransporter mRNA increased dramatically in Nhe1−/−parotid glands but not in those of Nhe2−/−orNhe3−/−mice, suggesting that compensation occurs for the loss of NHE1. The sodium content, chloride activity and osmolality of saliva in Nhe2−/−orNhe3−/−mice were comparable with those of wild-type mice. In contrast, Nhe1−/−mice displayed impaired NaCl absorption. These results suggest that in parotid duct cells apical NHE2 and NHE3 do not play a major role in Na+absorption. These results also demonstrate that basolateral NHE1 and apical NHE2 modulate saliva secretion in vivo, especially during sustained stimulation when secretion depends less on Na+/K+/2Cl−cotransporter activity.

1The Na+‐H+exchanger NHE3 and the thiazide‐sensitive Na+‐Cl−cotransporter NCC are the major apical sodium transporters in the proximal convoluted tubule and the distal convoluted tubule of the kidney, respectively. We investigated the mechanism of compensation that allows maintenance of sodium balance in NHE3 knockout mice and in NCC knockout mice.2We used a so‐called ‘targeted proteomics’ approach, which profiles the entire renal tubule with regard to changes in Na+transporter and aquaporin abundance in response to the gene deletions. Specific antibodies to the Na+transporters and aquaporins expressed along the nephron were utilized to determine the relative abundance of each transporter. Semiquantitative immunoblotting was used which gives an estimate of the percentage change in abundance of each transporter in knockout compared with wild‐type mice.3In NHE3 knockout mice three changes were identified which could compensate for the loss of NHE3‐mediated sodium absorption. (a) The proximal sodium‐phosphate cotransporter NaPi‐2 was markedly upregulated. (b) In the collecting duct, the 70 kDa form of the γ‐subunit of the epithelial sodium channel, ENaC, exhibited an increase in abundance. This is thought to be an aldosterone‐stimulated form of γ‐ENaC. (c) Glomerular filtration was significantly reduced.4In the NCC knockout mice, amongst all the sodium transporters expressed along the renal tubule, only the 70 kDa form of the γ‐subunit of the epithelial sodium channel, ENaC, exhibited an increase in abundance.5In conclusion, both mouse knockout models demonstrated successful compensation for loss of the deleted transporter. More extensive adaptation occurred in the case of the NHE3 knockout, presumably because NHE3 is responsible for much more sodium absorption in normal mice than in NCC knockout mice.

NHE3 is the predominant isoform responsible for apical membrane Na+/H+exchange in the proximal tubule. Deletion of NHE3 by gene targeting results in an NHE3−/−mouse with greatly reduced proximal tubuleHCO3−absorption compared with NHE3+/+animals (P. J. Schultheis, L. L. Clarke, P. Meneton, M. L. Miller, M. Soleimani, L. R. Gawenis, T. M. Riddle, J. J. Duffy, T. Doetschman, T. Wang, G. Giebisch, P. S. Aronson, J. N. Lorenz, and G. E. Shull.Nature Genet. 19: 282–285, 1998). The purpose of the present study was to evaluate the role of other acidification mechanisms in mediating the remaining component of proximal tubule HCO3−reabsorption in NHE3−/−mice. Proximal tubule transport was studied by in situ microperfusion. Net rates ofHCO3−(JHCO3) and fluid absorption (Jv) were reduced by 54 and 63%, respectively, in NHE3 null mice compared with controls. Addition of 100 μM ethylisopropylamiloride (EIPA) to the luminal perfusate caused significant inhibition ofJHCO3andJvin NHE3+/+mice but failed to inhibitJHCO3orJvin NHE3−/−mice, indicating lack of activity of NHE2 or other EIPA-sensitive NHE isoforms in the null mice. Addition of 1 μM bafilomycin caused a similar absolute decrement inJHCO3in wild-type and NHE3 null mice, indicating equivalent rates ofHCO3−absorption mediated by H+-ATPase. Addition of 10 μM Sch-28080 did not reduceJHCO3in either wild-type or NHE3 null mice, indicating lack of detectable H+-K+-ATPase activity in the proximal tubule. We conclude that, in the absence of NHE3, neither NHE2 nor any other EIPA-sensitive NHE isoform contributes to mediating HCO3−reabsorption in the proximal tubule. A significant component ofHCO3−reabsorption in the proximal tubule is mediated by bafilomycin-sensitive H+-ATPase, but its activity is not significantly upregulated in NHE3 null mice.

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−/−) and heterozygous (Nhe3+/−) knockout mice, and in their wild-type (WT,Nhe3+/+) littermates. Arterial pressure was lower in theNhe3−/−mice (89 ± 6 mmHg) compared withNhe3+/+(118 ± 4) andNhe3+/−(108 ± 5). Collections from proximal and distal tubules demonstrated that proximal fluid reabsorption was blunted in bothNhe3+/−andNhe3−/−mice (WT, 4.2 ± 0.3;Nhe3+/−, 3.4 ± 0.2; andNhe3−/−, 2.6 ± 0.3 nl/min; P< 0.05). However, distal delivery of fluid was not different among the three groups of mice (WT, 3.3 ± 0.4 nl/min;Nhe3+/−, 3.3 ± 0.2 nl/min; andNhe3−/−, 3.0 ± 0.4 nl/min; P< 0.05). InNhe3−/−mice, this compensation was largely attributable to decreased single-nephron glomerular filtration rate (SNGFR): 10.7 ± 0.9 nl/min in theNhe3+/+vs. 6.6 ± 0.8 nl/min in theNhe3−/−, measured distally. Proximal-distal SNGFR differences inNhe3−/−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 inNhe3−/−animals. In contrast toNhe3−/−mice, normalization of early distal flow rate inNhe3+/−mice was not related to decreased SNGFR (9.9 ± 0.7 nl/min), but rather, to increased fluid reabsorption in the loop segment (Nhe3+/+, 2.6 ± 0.2;Nhe3+/−, 3.6 ± 0.5 nl/min). We conclude that NHE3 is a major Na/H exchanger isoform mediating Na+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.

Mice with a targeted disruption of Na+/H+exchanger NHE-3 gene show significant reduction inHCO3−reabsorption in proximal tubule, consistent with the absence of NHE-3. SerumHCO3−, however, is only mildly decreased (P. Schulties, L. L. Clarke, P. Meneton, M. L. Miller, M. Soleimani, L. R. Gawenis, T. M. Riddle, J. J. Duffy, T. Doetschman, T. Wang, G. Giebisch, P. S. Aronson, J. N. Lorenz, and G. E. Shull.Nature Genet.19: 282–285, 1998), indicating possible adaptive upregulation ofHCO3−-absorbing transporters in collecting duct of NHE-3-deficient (NHE-3 −/−) mice. Cortical collecting duct (CCD) and outer medullary collecting duct (OMCD) were perfused, and total CO2(netHCO3−flux,JtCO2) was measured in the presence of 10 μM Schering 28080 (SCH, inhibitor of gastric H+-K+-ATPase) or 50 μM diethylestilbestrol (DES, inhibitor of H+-ATPase) in both mutant and wild-type (WT) animals. In CCD,JtCO2increased in NHE-3 mutant mice (3.42 ± 0.28 in WT to 5.71 ± 0.39 pmol ⋅ min−1⋅ mm tubule−1in mutants,P< 0.001). The SCH-sensitive netHCO3−flux remained unchanged, whereas the DES-sensitive HCO3−flux increased in the CCD of NHE-3 mutant animals. In OMCD,JtCO2increased in NHE-3 mutant mice (8.8 ± 0.7 in WT to 14.2 ± 0.6 pmol ⋅ min−1⋅ mm tubule−1in mutants,P< 0.001). Both the SCH-sensitive and the DES-sensitive HCO3−fluxes increased in the OMCD of NHE-3 mutant animals. Northern hybridizations demonstrated enhanced expression of the basolateral Cl−/HCO3−exchanger (AE-1) mRNA in the cortex. The gastric H+-K+-ATPase mRNA showed upregulation in the medulla but not the cortex of NHE-3 mutant mice. Our results indicate thatHCO3−reabsorption is enhanced in CCD and OMCD of NHE-3-deficient mice. In CCD, H+-ATPase, and in the OMCD, both H+-ATPase and gastric H+-K+-ATPase contribute to the enhanced compensatoryHCO3−reabsorption in NHE-3-deficient animals.

The onset of salivary gland fluid secretion in response to muscarinic stimulation is accompanied by up-regulation of Na+/H+exchanger (NHE) activity. Although multiple NHE isoforms (NHE1, NHE2, and NHE3) have been identified in salivary glands, little is known about their specific function(s) in resting and secreting acinar cells. Mice with targeted disruptions of the Nhe1, Nhe2, and Nhe3genes were used to investigate the contribution of these proteins to the stimulation-induced up-regulation of NHE activity in mouse parotid acinar cells. The lack of NHE1, but not NHE2 or NHE3, prevented intracellular pH recovery from an acid load in resting acinar cells, in acini stimulated to secrete with the muscarinic agonist carbachol, and in acini shrunken by hypertonic addition of sucrose. In HCO3−-containing solution, the rate of intracellular pH recovery from a muscarinic agonist-stimulated acid load was significantly inhibited in acinar cells from mice lacking NHE1, but not in cells from NHE2- or NHE3-deficient mice. These data demonstrate that NHE1 is the major regulator of intracellular pH in both resting and muscarinic agonist-stimulated acinar cells and suggest that up-regulation of NHE1 activity has an important role in modulating saliva production in vivo.

Multiple AE2 Cl-/HCO3-exchanger mRNAs have been identified in rat. To determine the genetic basis for these mRNAs and whether they encode different variants of the exchanger, we used both rapid amplification of cDNA ends and S1 nuclease protection protocols and examined the organization of the gene. mRNAs encoding three N-terminal variants of AE2 (AE2a, AE2b, and AE2c) were identified and shown to be transcribed from alternative promoters. The AE2a transcription unit consists of 23 exons, with exons 1 and 2 containing 5′-untranslated sequence and the first 17 codons. The first exon of AE2b is located in intron 2; it contains 5′-untranslated sequence and an alternative 3-amino acid N-terminal coding sequence and is spliced to exon 3. The first exon of AE2c is located in intron 5; it consists of 5′-untranslated sequence and is spliced to exon 6, which contains the translation initiation codon corresponding to Met-200 of AE2a. Northern analysis shows that AE2a is expressed in all tissues, AE2b exhibits a more restricted distribution with highest levels in stomach, and AE2c is expressed only in stomach. Thus, the use of alternative promoters leads to the production of three N-terminal variants of AE2 that exhibit tissue-specific patterns of expression.

1Cell‐specific reverse transcriptase‐polymerase chain reaction (RT‐PCR), immunolocalization and microspectrofluorometry were used to identify and localize the Na+‐H+exchanger (NHE) isoforms expressed in the submandibular gland (SMG) acinar and duct cells and their regulation by basolateral and luminal P2receptors in the duct.2The molecular and immunofluorescence analysis showed that SMG acinar and duct cells expressed NHE1 in the basolateral membrane (BLM). Duct cells also expressed NHE2 and NHE3 in the luminal membrane (LM).3Expression of NHE3 was unequivocally established by the absence of staining in SMG from NHE3 knockout mice. NHE3 was expressed in the LM and in subluminal regions of the duct.4Measurement of the inhibition of NHE activity by the amiloride analogue HOE 694 (HOE) suggested expression of NHE1‐like activity in the BLM and NHE2‐like activity in the LM of the SMG duct. Several acute and chronic treatments tested failed to activate NHE activity with low affinity for HOE as expected for NHE3. Hence, the physiological function and role of NHE3 in the SMG duct is not clear at present.5Activation of P2receptors resulted in activation of an NHE‐independent, luminal H+transport pathway that markedly and rapidly acidified the cells. This pathway could be blocked by luminal but not basolateral Ba2+.6Stimulation of P2Ureceptors expressed in the BLM activated largely NHE1‐like activity, and stimulation of P2Zreceptors expressed in the LM activated largely NHE2‐like activity.7The interrelation between basolateral and luminal NHE activities and their respective regulation by P2Uand P2Zreceptors can be used to co‐ordinate membrane transport events in the LM and BLM during active Na+reabsorption by the SMG duct.

Mutations in the gene encoding the thiazide-sensitive Na+-Cl−cotransporter (NCC) of the distal convoluted tubule cause Gitelman's syndrome, an inherited hypokalemic alkalosis with hypomagnesemia and hypocalciuria. These metabolic abnormalities are secondary to the deficit in NaCl reabsorption, but the underlying mechanisms are unclear. To gain a better understanding of the role of NCC in sodium and fluid volume homeostasis and in the pathogenesis of Gitelman's syndrome, we used gene targeting to prepare an NCC-deficient mouse. Null mutant (Ncc−/−) mice appear healthy and are normal with respect to acid-base balance, plasma electrolyte concentrations, serum aldosterone levels, and blood pressure.Ncc−/−mice retain Na+as well as wild-type mice when fed a Na+-depleted diet; however, after 2 weeks of Na+depletion the mean arterial blood pressure of Ncc−/−mice was significantly lower than that of wild-type mice. In addition, Ncc−/−mice exhibited increased renin mRNA levels in kidney, hypomagnesemia and hypocalciuria, and morphological changes in the distal convoluted tubule. These data indicate that the loss of NCC activity in the mouse causes only subtle perturbations of sodium and fluid volume homeostasis, but renal handling of Mg2+and Ca2+are altered, as observed in Gitelman's syndrome.

Genetic ablation of the NHE2 Na+/H+ exchanger causes gastric achlorhydria, absorptive defects in kidney and colon, and low fertility. Here we show that NHE2 is expressed in the pituitary, with the highest mRNA expression in pars distalis and lower expression in pars intermedia. In pars distalis of NHE2-null mice, prominent cyst-like dilatations of folliculo-stellate (FS) cell canaliculi developed with age, and there were increased FS cell area, accumulation of lipid in FS cell cytoplasm, redundancies in FS cell basement membrane, and other changes. The expansion of the canaliculi indicates that NHE2 is a major absorptive Na+/H+ exchanger in the luminal membranes lining the extensive network of channels formed by FS cells, which may provide a means of intrapituitary communication. The results suggest that NHE2 contributes to homeostatic regulation of the volume and composition of the canalicular fluid and may counter the secretory activity of the CFTR Cl− channel, which is known to be expressed in pituitary.