[Show abstract][Hide abstract] ABSTRACT: The majority of the Na(+) and Cl(-) filtered by the kidney is reabsorbed in the proximal tubule. In this nephron segment, a significant fraction of Cl(-) is transported via apical membrane Cl(-)-base exchange: Cl(-)-formate exchange, Cl(-)-oxalate exchange, Cl(-)-OH(-) exchange, and Cl(-)-HCO(3)(-) exchange. A search for the transporter responsible for apical membrane Cl(-)-formate exchange in the proximal tubule led to the identification of CFEX (SLC26A6). Functional expression studies in Xenopus oocytes demonstrated that CFEX is capable of mediating not only Cl(-)-formate exchange but also Cl(-)-oxalate exchange, Cl(-)-OH(-) exchange, and Cl(-)-HCO(3)(-) exchange. Studies in CFEX-null mice have begun to elucidate which of the anion exchange activities mediated by CFEX is important for renal physiology and pathophysiology in vivo. Measurements of transport in renal brush border vesicles isolated from CFEX-null mice demonstrated that CFEX primarily mediates Cl(-)-oxalate exchange rather than Cl(-)-formate exchange. Microperfusion studies in CFEX-null mice revealed that CFEX plays an essential role in mediating oxalate-dependent NaCl absorption in the proximal tubule. CFEX-null mice were found to have hyperoxaluria and a high incidence of calcium oxalate urolithiasis. The etiology of hyperoxaluria in CFEX-null mice was observed to be a defect in oxalate secretion in the intestine, leading to enhanced net absorption of ingested oxalate and elevation of plasma oxalate. Thus, by virtue of its function as a Cl(-)-oxalate exchanger, CFEX plays essential roles both in proximal tubule NaCl transport and in the prevention of hyperoxaluria and calcium oxalate nephrolithiasis.
Kidney International 11/2006; 70(7):1207-13. DOI:10.1038/sj.ki.5001741 · 8.56 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This study assessed the functional role of Na(+)/H(+) exchanger (NHE) isoforms NHE3 and NHE2 in the proximal tubule, loop of Henle, and distal convoluted tubule of the rat kidney by comparing sensitivity of transport to inhibition by Hoe-694 (an agent known to inhibit NHE2 but not NHE3) and S-3226 (an agent with much higher affinity for NHE3 than NHE2). Rates of transport of fluid (J(v)) and HCO(3)(-) (J(HCO3)) were studied by in situ microperfusion. In the proximal tubule, addition of ethylisopropylamiloride or S-3226 significantly reduced J(v) and J(HCO3), but addition of Hoe-694 caused no significant inhibition. In the loop of Henle, J(HCO3) was also inhibited by S-3226 and not by Hoe-694, although much higher concentrations of S-3226 were required than what was necessary to inhibit transport in the proximal tubule. In contrast, in the distal convoluted tubule, J(HCO3) was inhibited by Hoe-694 but not by S-3226. These results are consistent with the conclusion that NHE2 rather than NHE3 is the predominant isoform responsible for apical membrane Na(+)/H(+) exchange in the distal convoluted tubule, whereas NHE3 is the predominant apical isoform in the proximal tubule and possibly also in the loop of Henle.
American journal of physiology. Renal physiology 01/2002; 281(6):F1117-22. · 3.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In an attempt to identify proteins that assemble with the apical membrane Na(+)-H(+) exchanger isoform NHE3, we generated monoclonal antibodies (mAbs) against affinity-purified NHE3 protein complexes isolated from solubilized renal microvillus membrane vesicles. Hybridomas were selected based on their ability to immunoprecipitate NHE3. We have characterized in detail one of the mAbs (1D11) that specifically co-precipitated NHE3 but not villin or NaPi-2. Western blot analyses of microvillus membranes and immunoelectron microscopy of kidney sections showed that mAb 1D11 recognizes a 110-kDa protein highly expressed on the apical membrane of proximal tubule cells. Immunoaffinity chromatography was used to isolate the antigen against which mAb 1D11 is directed. N-terminal sequencing of the purified protein identified it as dipeptidyl peptidase IV (DPPIV) (EC ), which was confirmed by assays of DPPIV enzyme activity. We also evaluated the distribution of the NHE3-DPPIV complex in microdomains of rabbit renal brush border. In contrast to the previously described NHE3-megalin complex, which principally resides in a dense membrane population (coated pits) in which NHE3 is inactive, the NHE3-DPPIV complex was predominantly in the microvillar fraction in which NHE3 is active. Serial precipitation experiments confirmed that anti-megalin and anti-DPPIV antibodies co-precipitate different pools of NHE3. Taken together, these studies revealed an unexpected association of the brush border Na(+)-H(+) exchanger NHE3 with dipeptidyl peptidase IV in the proximal tubule. These findings raise the possibility that association with DPPIV may affect NHE3 surface expression and/or activity.
[Show abstract][Hide abstract] ABSTRACT: The absorption of NaCl in the proximal tubule is markedly stimulated by formate. This stimulation of NaCl transport is consistent with a cell model involving Cl(-)-formate exchange in parallel with pH-coupled formate recycling due to nonionic diffusion of formic acid or H(+)-formate cotransport. The formate recycling process requires H(+) secretion. Although Na(+)-H(+) exchanger isoform NHE3 accounts for the largest component of H(+) secretion in the proximal tubule, 40-50% of the rates of HCO absorption or cellular H(+) extrusion persist in NHE3 null mice. The purpose of the present investigation is to use NHE3 null mice to directly test the role of apical membrane NHE3 in mediating NaCl absorption stimulated by formate. We demonstrate that formate stimulates NaCl absorption in the mouse proximal tubule microperfused in vivo, but the component of NaCl absorption stimulated by formate is absent in NHE3 null mice. In contrast, stimulation of NaCl absorption by oxalate is preserved in NHE3 null mice, indicating that oxalate-stimulated NaCl absorption is independent of Na(+)-H(+) exchange. The virtually complete dependence of formate-induced NaCl absorption on NHE3 activity raises the possibility that NHE3 and the formate transporters are functionally coupled in the brush border membrane.
American journal of physiology. Renal physiology 09/2001; 281(2):F288-92. · 3.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A key function of the proximal tubule is retrieval of most of the vast quantities of NaCl and water filtered by the kidney. Physiological studies using brush border vesicles and perfused tubules have indicated that a major fraction of Cl(-) reabsorption across the apical membrane of proximal tubule cells occurs via Cl(-)-formate exchange. The molecular identity of the transporter responsible for renal brush border Cl(-)-formate exchange has yet to be elucidated. As a strategy to identify one or more anion exchangers responsible for mediating Cl(-) reabsorption in the proximal tubule, we screened the expressed sequence tag database for homologs of pendrin, a transporter previously shown to mediate Cl(-)-formate exchange. We now report the cDNA cloning of CFEX, a mouse pendrin homolog with expression in the kidney by Northern analysis. Sequence analysis indicated that CFEX very likely represents the mouse ortholog of human SLC26A6. Immunolocalization studies detected expression of CFEX, but not pendrin, on the brush border membrane of proximal tubule cells. Functional expression studies in Xenopus oocytes demonstrated that CFEX mediates Cl(-)-formate exchange. Taken together, these observations identify CFEX as a prime candidate to mediate Cl(-)-formate exchange in the proximal tubule and thereby to contribute importantly to renal NaCl reabsorption. Given its wide tissue distribution, CFEX also may contribute to transcellular Cl(-) transport in additional epithelia such as the pancreas and contribute to transmembrane Cl(-) transport in nonepithelial tissues such as the heart.
Proceedings of the National Academy of Sciences 08/2001; 98(16):9425-30. DOI:10.1073/pnas.141241098 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have previously shown that Na(+)-H(+) exchanger isoform NHE3 exists as both 9.6 and 21 S (megalin-associated) oligomers in the renal brush border. To characterize the oligomeric forms of the renal brush border Na(+)-H(+) exchanger in more detail, we performed membrane fractionation studies. We found that similar amounts of NHE3 were present in microvilli and a nonmicrovillar membrane domain of high density (dense vesicles). Horseradish peroxidase-labeled endosomes were not prevalent in the dense membrane fraction. However, megalin, which localizes primarily to the intermicrovillar microdomain of the brush border, was enriched in the dense vesicles, implicating this microdomain as the likely source of these membranes. Immunolocalization of NHE3 confirmed that a major fraction of the transporter colocalized with megalin in the intermicrovillar region of the brush border. Immunoprecipitation studies demonstrated that in microvilli the majority of NHE3 was not bound to megalin, while in the dense vesicles most of the NHE3 coprecipitated with megalin. Moreover, sucrose velocity gradient centrifugation experiments revealed that most NHE3 in microvilli sedimented with an S value of 9.6, while the S value of NHE3 in dense vesicles was 21. Finally, we examined the functional state of NHE3 in both membrane fractions. As assayed by changes in acridine orange fluorescence, imposing an outwardly directed Na(+) gradient caused generation of an inside acid pH gradient in the microvilli, indicating Na(+)-H(+) exchange activity, but not in the dense vesicles. Taken together, these data demonstrate that renal brush border NHE3 exists in two oligomeric states: a 9.6 S active form present in microvilli and a 21 S, megalin-associated, inactive form in the intermicrovillar microdomain of the apical plasma membrane. Thus, regulation of renal brush border Na(+)-H(+) exchange activity may be mediated by shifting the distribution between these forms of NHE3.
[Show abstract][Hide abstract] ABSTRACT: Na+/H+ 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 videomicroscopic 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+/H+ 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.
[Show abstract][Hide abstract] ABSTRACT: Kidney-specific cadherin (Ksp-cadherin, cadherin 16) is a novel, kidney-specific member of the cadherin superfamily that is expressed exclusively in the basolateral membrane of renal tubular epithelial cells. To characterize the Ksp-cadherin gene promoter, a lambda bacteriophage clone containing 3.7 kb of the proximal 5' flanking region of the mouse Ksp-cadherin gene was isolated. The transcription initiation site was mapped by RNase protection assays and 5' rapid amplification of cDNA ends, and a 709-bp intron was identified within the 5' untranslated region. The proximal 5' flanking region was "TATA-less" but contained other consensus promoter elements including an initiator (Inr), GC boxes, and a CAAT box. Potential binding sites were identified for transcription factors that are involved in tissue-specific gene expression including activator protein-2 (AP-2), hepatocyte nuclear factor-3 (HNF-3), basic helix-loop-helix (bHLH) proteins, CCAAT/enhancer-binding protein (C/EBP), and GATA factors. Transfection of luciferase reporter plasmids containing 2.6 kb of the 5' flanking region markedly increased luciferase activity in renal epithelial cells (MDCK and mIMCD-3) but not in mesenchymal cells (NIH 3T3 and MMR1). Deletion analysis identified an 82-bp region from -31 to -113 that was essential for promoter activity in transfected renal epithelial cells. Electrophoretic mobility-shift assays showed that mIMCD-3 cells contain nuclear proteins that bind to this region of the promoter. Mutational analysis showed that sequences within the HNF-3 consensus site and CAAT box were involved in protein binding and promoter activity. We conclude that the proximal 5' flanking region of the mouse Ksp-cadherin gene contains an orientation-dependent promoter that is kidney epithelial cell specific. The region of the promoter from -113 to -31 is required for transcriptional activity and contains binding sites for nuclear proteins that are specifically expressed in renal epithelial cells.
The American journal of physiology 11/1999; 277(4 Pt 2):F587-98. · 3.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Kidney-specific cadherin (Ksp-cadherin, cadherin 16) is a tissue-specific member of the cadherin superfamily that is expressed exclusively in the basolateral membrane of tubular epithelial cells in the kidney. To determine the basis for tissue-specific expression of Ksp-cadherin in vivo, we evaluated the activity of the promoter in transgenic mice. Transgenic mice containing 3.3 kb of the mouse Ksp-cadherin promoter and an Escherichia coli lacZ reporter gene were generated by pronuclear microinjection. Assays of beta-galactosidase enzyme activity showed that the transgene was expressed exclusively in the kidney in both adult and developing mice. Within the kidney, the transgene was expressed in a subset of renal tubular epithelial cells that endogenously expressed Ksp-cadherin and that were identified as collecting ducts by colabeling with Dolichos biflorus agglutinin. In the developing metanephros, expression of the transgene in the branching ureteric bud correlated with the developmental expression of Ksp-cadherin. Identical patterns of expression were observed in multiple founder mice, indicating that kidney specificity was independent of transgene integration site. However, heterocellular expression was observed consistent with repeat-induced gene silencing. We conclude that the Ksp-cadherin gene promoter directs kidney-specific expression in vivo. Regulatory elements that are sufficient to recapitulate the tissue- and differentiation-specific expression of Ksp-cadherin in the renal collecting duct are located within 3.3 kb upstream to the transcriptional start site.
The American journal of physiology 11/1999; 277(4 Pt 2):F599-610. · 3.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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 tubule HCO(-)(3) 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 HCO(-)(3) reabsorption in NHE3(-/-) mice. Proximal tubule transport was studied by in situ microperfusion. Net rates of HCO(-)(3) (J(HCO3)) and fluid absorption (J(v)) were reduced by 54 and 63%, respectively, in NHE3 null mice compared with controls. Addition of 100 microM ethylisopropylamiloride (EIPA) to the luminal perfusate caused significant inhibition of J(HCO3) and J(v) in NHE3(+/+) mice but failed to inhibit J(HCO3) or J(v) in NHE3(-/-) mice, indicating lack of activity of NHE2 or other EIPA-sensitive NHE isoforms in the null mice. Addition of 1 microM bafilomycin caused a similar absolute decrement in J(HCO3) in wild-type and NHE3 null mice, indicating equivalent rates of HCO(-)(3) absorption mediated by H(+)-ATPase. Addition of 10 microM Sch-28080 did not reduce J(HCO3) in 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 HCO(-)(3) reabsorption in the proximal tubule. A significant component of HCO(-)(3) reabsorption in the proximal tubule is mediated by bafilomycin-sensitive H(+)-ATPase, but its activity is not significantly upregulated in NHE3 null mice.
The American journal of physiology 09/1999; 277(2 Pt 2):F298-302. · 3.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The potential for Ksp-cadherin involvement in either the development or maintenance of the metanephric kidney was assessed by immunocytochemical localization of a monoclonal antibody directed against the rabbit isoform of Ksp-cadherin in both neonatal and adult rabbit kidneys. In the adult kidney Ksp-cadherin expression was detected on the basolateral membrane of all cell types in both the tubular nephron and the collecting system. Immunoelectron microscopy indicated that Ksp-cadherin was expressed at uniform levels along the entire length of both the lateral membranes and the basal infoldings of all tubular epithelial cell types. In the nephrogenic zone of the neonatal rabbit kidney Ksp-cadherin expression was detected exclusively on the basolateral membranes of epithelial cells in the more highly differentiated regions of the expanding ureteric duct. In the highly differentiated corticomedullary and medullary regions of the neonatal kidney, distinct basolateral staining was observed in all segments of the tubular nephron and the collecting system. The relatively late appearance of Ksp-cadherin expression in the developing metanephros indicates that Ksp-cadherin probably does not participate in the direction of renal morphogenesis. However, the high levels of Ksp-cadherin expression observed in all segments of the tubular nephron and the collecting system in the adult kidney suggests that it may play a role in the maintenance of the terminally differentiated tubular epithelial phenotype.
The American journal of physiology 08/1999; 277(1 Pt 2):F146-56. · 3.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We investigated whether the renal brush border Na+/H+ exchanger NHE3 exists in assemblies with other proteins in native kidney membranes. To this end we generated monoclonal antibodies (mAbs) against affinity purified NHE3 protein complexes. Hybridomas were selected based on ability to immunoprecipitate NHE3. One of the resulting mAbs (10A3) labeled a high molecular mass (>200 kDa) protein and stained primarily the coated pit region of the proximal tubule in a manner similar to that described for megalin (gp330). We then confirmed that both mAb 10A3 and a known anti-megalin mAb immunoprecipitated and immunoblotted the same protein, namely megalin. mAb 10A3 specifically co-precipitated NHE3 but not villin or NaPi-2 from solubilized renal membranes, indicating specificity of the NHE3-megalin interaction. When immunoprecipitations were performed using either 10A3 or anti-NHE3 mAb 2B9 after separation of solubilized renal proteins by sucrose velocity gradient centrifugation, we found that NHE3 exists in two states with distinct sedimentation coefficients, a 9.6 S megalin-free form and a 21 S megalin-bound form, and that when NHE3 assembles with megalin, epitopes within the carboxyl-terminal 131 amino acids of NHE3 are blocked. Taken together, these findings indicate that a significant pool of NHE3 exists as a multimeric complex with megalin in the brush border of the proximal tubule.
[Show abstract][Hide abstract] ABSTRACT: Mammalian Na+/H+ exchangers (NHEs) are a family of transport proteins (NHE1-NHE5). To date, the cellular and subcellular localization of NHE4 has not been characterized using immunochemical techniques. We purified a fusion protein containing a portion of rat NHE4 (amino acids 565-675) to use as immunogen. A monoclonal antibody (11H11) was selected by ELISA. It reacted specifically with both the fusion protein and to a 60- to 65-kDa polypeptide expressed in NHE4-transfected LAP1 cells. By Western blot analysis, NHE4 was identified as a 65- to 70-kDa protein that was expressed most abundantly in stomach and in multiple additional epithelial and nonepithelial rat tissues including skeletal muscle, heart, kidney, uterus, and liver. Subcellular localization of NHE4 in the kidney was evaluated by Western blot analysis of membrane fractions isolated by Percoll gradient centrifugation. NHE4 was found to cofractionate with the basolateral markers NHE1 and Na+-K+-ATPase rather than the luminal marker gamma-glutamyl transferase. In stomach, NHE4 was detected by immunoperoxidase labeling on the basolateral membrane of cells at the base of the gastric gland. We conclude that NHE4 is a 65- to 70-kDa protein with a broad tissue distribution. In two types of epithelial cells, kidney and stomach, NHE4 is localized to the basolateral membrane.
The American journal of physiology 11/1998; 275(4 Pt 2):F510-7. · 3.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ksp-cadherin is a novel kidney-specific member of the cadherin superfamily of cell adhesion molecules. We have determined the complete cDNA coding sequences of both the human and the mouse isoforms of Ksp-cadherin. The inferred amino acid sequences of the human and mouse isoforms are 79 and 75% identical to the originally described rabbit isoform of Ksp-cadherin (Thomson et al., 1995; J. Biol. Chem. 270, 17594-17601), respectively. The relative locations of cadherin-specific sequence motifs, putative N-glycosylation sites, and characteristic protein domains are entirely conserved in all three isoforms. Multiple organ Northern analyses indicate that, as in the rabbit, both the human and the mouse Ksp-cadherin transcripts appear to have distinct kidney-specific distributions. The human Ksp-cadherin gene (CDH16) maps to chromosome 16q21-proximal 16q22. The mouse Ksp-cadherin gene (Cdh16) was localized to a highly syntenic region of distal Chromosome 8. Both the human and the mouse Ksp-cadherin genes were localized to previously identified clusters of cadherin gene sequences, consistent with the hypothesis that most cadherin family members arose by gene duplication from a single ancestral gene at a relatively early stage in the evolution of the mammalian genome.
[Show abstract][Hide abstract] ABSTRACT: In metabolic acidosis, the capacity of the proximal tubule for bicarbonate absorption is enhanced, whereas NaCl reabsorption is inhibited. Recent evidence indicates that transcellular NaCl absorption in the proximal tubule is mediated by apical membrane Cl/formate exchange and Cl/oxalate exchange, in parallel with recycling of these organic anions. We evaluated whether the effect of metabolic acidosis to inhibit NaCl reabsorption in the proximal tubule is due at least in part to inhibition of organic anion-dependent NaCl transport in this nephron segment. Absorption rates of bicarbonate (JHCO3), chloride (JCl), and fluid (Jv) were measured in rat proximal tubule segments microperfused in situ. We confirmed that metabolic acidosis stimulates JHCO3 in tubules microperfused with 25 mM HCO3, pH 7.4. For measurements of JCl, tubules were microperfused with a low-bicarbonate (5 mM), high-chloride solution, simulating conditions in the late proximal tubule. Under these conditions, baseline JCl and Jv measured in the absence of formate and oxalate were not significantly different between control and acidotic rats. However, whereas addition of 50 ¿M formate or 1 ¿M oxalate to luminal and capillary perfusates markedly stimulated JCl and Jv in control rats, formate and oxalate failed to stimulate JCl and Jv in acidotic rats. We conclude that metabolic acidosis markedly downregulates organic anion-stimulated NaCl absorption, thereby allowing differential regulation of proximal tubule NaHCO3 and NaCl transport.
The American journal of physiology 07/1998; 274(6 Pt 2):F1015-9. · 3.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Increased peripheral blood cell Na-H exchange (NHE) and erythrocyte Na-Li countertransport activity have been reported in hypertension and diabetic nephropathy and correlated with increased activity of the renal brush border Na-H exchanger. A relationship between cation exchange activities of blood cells and renal brush border membranes might exist if both were mediated by the same NHE isoform. We generated isoform-specific antibodies to compare the expression of NHE1 and NHE3 in peripheral blood cell membranes and renal cortical membrane vesicles. An NHE1-specific monoclonal antibody reacted with a 199- to 110-kD protein in basolateral membrane fractions isolated from rabbit and rat kidney. NHE1 protein expression was also detected in erythrocytes, platelets, and lymphocytes isolated from rabbit and rat. Two polyclonal antisera generated against nonoverlapping portions of NHE3 reacted with proteins of 82 and 85 kD in brush border membrane fractions isolated from rabbit and rat kidney, respectively, but failed to detect NHE3 expression in blood cells. These data do not support the hypothesis that Na-H exchanger of Na-Li countertransport in blood cells takes place via the renal brush border membrane NHE isoform, namely NHE3.
[Show abstract][Hide abstract] ABSTRACT: Previous immunochemical studies have shown that NHE3 is an apical Na+/H+ exchanger in some renal epithelia. The purpose of the present study was to develop high-affinity, isoform-specific monoclonal antibodies (MAbs) that would be useful for carrying out high-resolution immunocytochemical studies of NHE3 in the adult and neonatal mammalian kidney. Three MAbs were developed to a fusion protein containing amino acids 702-832 of rabbit NHE3. Specificity was established by immunoblotting membranes from NHE-deficient LAP cells that had been transfected with either NHE1,-2, -3, or -4. With the use of high-resolution immunocytochemical techniques, NHE3 was found in vesicles in the apical cytoplasm of proximal tubule cells, as well as in the apical plasma membrane of the proximal tubule, and in both the thin and thick limbs of the loop of Henle. When localized in the 1-day-old rat kidney, NHE3 was first detected in the late stages of the S-shaped body. In later stages of nephron development, the pattern of NHE3 staining was similar to that seen in the adult. This study demonstrates 1) the specificity of three MAbs for Na+/H+ exchanger isoform NHE3; 2) NHE3 is present in an intracellular vesicular compartment in cells of the proximal tubule, consistent with possible regulation by membrane recycling; and 3) NHE3 is expressed on the apical membrane in early stages of the developing nephron.
The American journal of physiology 09/1997; 273(2 Pt 2):F289-99. · 3.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The major fraction of filtered Cl- is reabsorbed in the proximal tubule. An important component of Cl- reabsorption is passive and paracellular, driven by the lumen-negative potential difference in the early proximal tubule and the outwardly directed concentration gradient for Cl- in the later proximal tubule. Evidence suggests that a significant additional component of NaCl reabsorption in the proximal tubule is active and transcellular. Cl-/formate and Cl-/oxalate exchangers have been identified as mechanisms of uphill Cl- entry across the apical membrane. For steady-state Cl- absorption to occur by these mechanisms, formate and oxalate must recycle from lumen to cell. Recent studies indicate that recycling of formate occurs by H(+)-coupled formate transport in parallel with Na+/H+ exchange, whereas oxalate recycling takes place by oxalate/sulfate exchange in parallel with Na(+)-sulfate cotransport. The predominant route for Cl- exit across the basolateral membrane is via Cl- channels. Unresolved issues include the adequacy of formate recycling to sustain Cl- absorption by Cl-/formate exchange, the existence and contributions of additional mechanisms for apical Cl entry and basolateral Cl- exit, and the relative magnitudes of transcellular and paracellular transport under physiological conditions. In addition, the molecular identification and mechanisms of regulation of the Cl-/formate and Cl-/oxalate exchangers remain to be defined.
The American journal of physiology 09/1997; 273(2 Pt 2):F179-92. · 3.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have studied the mechanisms of NaCl transport in the mammalian proximal tubule. We identified Cl(-)-formate and Cl(-)-oxalate exchangers as possible mechanism's of uphill Cl- entry across the apical membrane of proximal tubule cells. For steady state Cl- absorption to occur by these mechanisms, formate and oxalate must recycle from lumen to cell. Recycling of formate from lumen to cell may occur by H(+)-coupled formate transport and nonionic diffusion of formic acid in parallel with Na(+)-H+ exchange. Oxalate recycling from lumen to cell may take place by oxalate-sulfate exchange in parallel with Na(+)-sulfate cotransport. Cl- exit across the basolateral membrane is most likely mediated by Cl- channels. To identify the Na(+)-H+ exchanger (NHE) isoform(s) expressed on the brush border membrane of proximal tubule cells, we developed isoform-specific monoclonal and polyclonal antibodies. We found that NHE1 is present on the basolateral membrane of all nephron segments, whereas NHE3 is present on the apical membrane of cells in the proximal tubule and the thin and thick limbs of the loop of Henle. NHE3 is also present in a population of subapical intracellular vesicles, suggesting possible regulation by membrane trafficking. The inhibitor sensitivity of Na(+)-H+ exchange in renal brush border vesicles indicates that it is mediated by NHE3 under baseline conditions and during up-regulation by metabolic acidosis. Increased apical membrane Na(+)-H+ exchange activity in response to metabolic acidosis and during renal maturation is associated with increased NHE3 protein expression. Finally, we found that the organic anion-dependent absorption of Cl- is markedly down-regulated in metabolic acidosis in parallel with the up-regulation of brush border membrane Na(+)-H+ exchange. Thus, differential regulation of apical membrane ion exchangers may provide a mechanism to regulate the relative rates of NaHCO3 and NaCl reabsorption.
Wiener klinische Wochenschrift 07/1997; 109(12-13):435-40. · 0.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The aims of the present study were to estimate the fraction of renal brush border membrane Na+-H+ exchange activity mediated by the isoform NHE3 and to evaluate whether the increased brush border Na+-H+ exchange observed in metabolic acidosis is due to increased expression of NHE3 protein. Compared with other isoforms, NHE3 is known to have a unique profile of sensitivity to pharmacologic inhibitors, including relative resistance to amiloride analogs and HOE694. We therefore assessed the inhibitor sensitivity of pH gradient-stimulated 22Na uptake in renal brush border vesicles isolated from normal rats. The I50 values for amiloride (30 microM), dimethylamiloride (10 microM), ethylisopropylamiloride (6 microM), and HOE694 (>100 microM) were markedly dissimilar from those reported for NHE1 and NHE2 but were nearly identical to reported values for NHE3. Na+-H+ exchange activity in renal brush border vesicles isolated from rats with 5 days of NH4Cl-induced metabolic acidosis was increased 1.5-fold compared with control rats, with no change in inhibitor sensitivity. Western blot analysis indicated that NHE3 protein expression was greater in brush border membranes from acidotic compared with control rats. We conclude that virtually all measured Na+-H+ exchange activity in brush border membranes from control and acidotic rats is mediated by NHE3 and that metabolic acidosis causes increased expression of renal brush border NHE3 protein.