MAB3136 Sigma-AldrichAnti-Na+/H+ Exchanger-3 Antibody, isoform NHE3, clone 4F5

A major mechanism of electroneutral NaCl absorption in the human ileum and colon involves coupling of Na(+)/H(+) and Cl(-)/HCO(3)(-) exchangers. Disturbances in these mechanisms have been implicated in diarrheal conditions. Probiotics such as Lactobacillus have been indicated to be beneficial in the management of gastrointestinal disorders, including diarrhea. However, the molecular mechanisms underlying antidiarrheal effects of probiotics have not been fully understood. We have previously demonstrated Lactobacillus acidophilus (LA) to stimulate Cl(-)/HCO3- exchange activity via an increase in the surface levels and expression of the Cl(-)/HCO3- exchanger DRA in vitro and in vivo. However, the effects of LA on NHE3, the Na(+)/H(+) exchanger involved in the coupled electroneutral NaCl absorption, are not known. Current studies were, therefore, undertaken to investigate the effects of LA on the function and expression of NHE3 and to determine the mechanisms involved. Treatment of Caco2 cells with LA or its conditioned culture supernatant (CS) for 8-24 h resulted in a significant increase in Na(+)/H(+) exchange activity, mRNA, and protein levels of NHE3. LA-CS upregulation of NHE3 function and expression was also observed in SK-CO15 cells, a human colonic adenocarcinoma cell line. Additionally, LA treatment increased NHE3 promoter activity, suggesting involvement of transcriptional mechanisms. In vivo, mice gavaged with live LA showed significant increase in NHE3 mRNA and protein expression in the ileum and colonic regions. In conclusion, LA-induced increase in NHE3 expression may contribute to the upregulation of intestinal electrolyte absorption and might underlie the potential antidiarrheal effects of probiotics.

Expression of a cytosolic cyan fluorescent fusion protein of angiotensin II (ECFP/ANG II) in proximal tubules increases blood pressure in rodents. To determine cellular signaling pathways responsible for this response, we expressed ECFP/ANG II in transport-competent mouse proximal convoluted tubule cells (mPCT) from wild-type (WT) and type 1a ANG II receptor-deficient (AT(1a)-KO) mice and measured its effects on intracellular ANG II levels, surrogates of Na/H exchanger 3 (NHE3)-dependent Na(+) absorption, as well as MAP kinases and NF-κB signaling. In WT mPCT cells, ECFP/ANG II expression doubled ANG II levels, increased NHE3 expression and membrane phospho-NHE3 proteins threefold and intracellular Na(+) concentration by 65%. These responses were associated with threefold increases in phospho-ERK 1/2 and phospho-p38 MAPK, fivefold increases in p65 subunit of NF-κB, and threefold increases in phospho-IKKα/β (Ser 176/180) proteins. These signaling responses to ECFP/ANG II were inhibited by losartan (AT(1) blocker), PD123319 (AT(2) blocker), U0126 (MEK1/MEK2 inhibitor), and RO 106-9920 (NF-κB inhibitor). In mPCT cells of AT(1a)-KO mice, ECFP/ANG II also increased the levels of NHE3, p-ERK1/2, and p65 proteins above their controls, but considerably less so than in WT cells. In WT mice, selective expression of ECFP/ANG II in vivo in proximal tubules significantly increased blood pressure and indices of sodium reabsorption, in particular levels of phosphorylated NHE3 protein in the membrane fraction and proton gradient-stimulated (22)Na(+) uptake by proximal tubules. We conclude that intracellular ANG II may induce NHE3 expression and activation in mPCTs via AT(1a)- and AT(2) receptor-mediated activation of MAP kinases ERK 1/2 and NF-κB signaling pathways.

Angiotensin (ANG) II via AT1 receptors (AT1Rs) maintains sodium homeostasis by regulating renal sodium transporters including Na(+)/H(+) exchanger 3 (NHE3) in a biphasic manner. Low-ANG II concentration stimulates whereas high concentrations inhibit NHE3 activity. Oxidative stress has been shown to upregulate AT1R function that could modulate the ANG II-mediated NHE3 regulation. This study was designed to identify the signaling pathways responsible for ANG II-mediated biphasic regulation of proximal tubular NHE3 and the effect of oxidative stress on this phenomenon. Male Sprague-Dawley rats were chronically treated with a pro-oxidant L-buthionine sulfoximine (BSO) with and without an antioxidant tempol in tap water for 3 wk. BSO-treated rats exhibited oxidative stress and high blood pressure. At low concentration (1 pM) ANG II increased NHE3 activity in proximal tubules from all animals. However, in BSO-treated rats, the stimulation was more robust and was normalized by tempol treatment. ANG II (1 pM)-mediated NHE3 activation was abolished by AT1R blocker, intracellular Ca(2+) chelator, and inhibitors of phospholipase C (PLC) and Ca(2+)-dependent calmodulin (CaM) but it was insensitive to Giα and protein kinase C inhibitors or AT2R antagonist. A high concentration of ANG II (1 μM) inhibited NHE3 activity in control and tempol-treated rats. However, in BSO-treated rats, ANG II (1 μM) continued to induce NHE3 stimulation. Tempol restored the inhibitory effect of ANG II (1 μM) in BSO-treated rats. The inhibitory effect of ANG II (1 μM) involved AT1R-dependent, cGMP-dependent protein kinase (PKG) activation and was independent of AT2 receptor and nitric oxide signaling. We conclude that ANG II stimulates NHE3 via AT1R-PLC-CaM pathway and inhibits NHE3 by AT1R-PKG activation. Oxidative stress impaired ANG II-mediated NHE3 biphasic response in that stimulation was observed at both high- and low-ANG II concentration.

We studied the distribution of Na(+)/HCO(3)(-) cotransporters (NBC) and Na(+)/H(+) exchanger type 3 (NHE3) in the laryngeal nerve by immunohistochemistry to elucidate the spatial relationship of pH regulation system in the peripheral nerves. The nervous components, i.e., the nerve cells in the nodose and local ganglia and nerve fibers, were immunoreactive for NBC. Glial components such as Schwann cells and satellite cells surrounding nerve fibers and nerve cell bodies were also immunoreactive for NBC in most cases, while the cells comprising the perineurium and endoneurium were immunoreactive for NHE3. These results suggest that NBC-dependent pH regulation systems are present in the laryngeal nerve. Whereas, NHE3 may regulate extracellular pH rather than intracellular pH. In conclusion, spatial relationship of NBC and NHE3 in the laryngeal nerve would be important for pH regulation. Perineural lymph may have key role for acid-induced modulation of axons and Schwann cells.

This study compared the expression of enzymes and transport and channel proteins involved in the regulation of sodium reabsorption in the kidney of Dahl salt-sensitive (DS) and salt-resistant Brown-Norway (BN) and consomic rats (SS.BN13), in which chromosome 13 from the BN rat has been introgressed into the DS genetic background. The expression of the Na+/K+/2Cl- (BSC-1) cotransporter, Na+/H+ exchanger (NHE3), and Na+-K+-ATPase proteins were similar in the renal cortex of DS, BN, and SS.BN13 rats fed either a low-salt (0.1% NaCl) or a high-salt (8% NaCl) diet. The expression of the BSC-1 and the renal outer medullary K+ channel (ROMK) were higher, whereas the expression of the cytochrome P4504A proteins responsible for the formation of 20-hydroxyeicosatetraenoic (20-HETE) was lower in the outer medulla of the kidney of DS than in BN or SS.BN13 rats fed either a low-salt or a high-salt diet. In addition, the renal formation and excretion of 20-HETE was lower in DS than in BN and SS.BN13 rats. These results suggest that overexpression of ROMK and BSC-1 in the thick ascending limb combined with a deficiency in renal formation of 20-HETE may predispose Dahl S rats fed a high-salt diet to Na+ retention and hypertension.

This study examined the effects of chronic blockade of the renal formation of epoxyeicosatrienoic acids and 20-hydroxyeicosatetraenoic acid with 1-aminobenzotriazole (ABT; 50 mg.kg(-1). day(-1) ip for 5 days) on pressure natriuresis and the inhibitory effects of elevations in renal perfusion pressure (RPP) on Na(+)-K(+)-ATPase activity and the distribution of the sodium/hydrogen exchanger (NHE)-3 in the proximal tubule of rats. In control rats (n = 15), sodium excretion rose from 2.3 +/- 0.4 to 19.4 +/- 1.8 microeq.min(-1).g kidney weight(-1) when RPP was increased from 114 +/- 1 to 156 +/- 2 mmHg. Fractional excretion of lithium rose from 28 +/- 3 to 43 +/- 3% of the filtered load. Chronic treatment of the rats with ABT for 5 days (n = 8) blunted the natriuretic response to elevations in RPP by 75% and attenuated the increase in fractional excretion of lithium by 45%. In vehicle-treated rats, renal Na(+)-K(+)-ATPase activity fell from 31 +/- 5 to 19 +/- 2 micromol P(i).mg protein(-1).h(-1) and NHE-3 protein was internalized from the brush border of the proximal tubule after an elevation in RPP. In contrast, Na(+)-K(+)-ATPase activity and the distribution of NHE-3 protein remained unaltered in rats treated with ABT. These results suggest that cytochrome P-450 metabolites of arachidonic acid contribute to pressure natriuresis by inhibiting Na(+)-K(+)-ATPase activity and promoting internalization of NHE-3 protein from the brush border of the proximal tubule.

The efferent ducts reabsorb most of the fluid released with spermatozoa from the testis. This absorptive capacity results in a severalfold increase in sperm concentration in the proximal epididymis and is partly responsible for maintenance of the optimal microenvironment for the sperm maturation. The fluid absorption is coupled to active Na+ transport and is inhibitable by amiloride, both of which suggest a role for a Na+/H+ exchanger (NHE). NHE3 is an apical membrane NHE responsible for sodium absorption in renal proximal tubule and intestinal epithelium. In the present study, we examined the expression of NHE3 messenger RNA (mRNA) and protein in the rat efferent ducts by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting and the localization of NHE3 by indirect immunofluoresce. RT-PCR indicated the expression of NHE3 mRNA, and Western blotting showed an NHE3 protein in the efferent duct membrane homogenate. By immunofluorescence, NHE3 was localized to the apical membrane of the nonciliated cells in the efferent duct epithelium, which also expressed aquaporin-1 water channel protein. These results suggest that NHE3 potentially plays an important role in the fluid reabsorption in the efferent ducts.

In the male reproductive tract, the epididymis plays an important role in mediating transepithelial bicarbonate transport and luminal acidification. In the proximal vas deferens, a significant component of luminal acidification is Na+-independent, and mediated by specific cells that possess apical vacuolar proton pumps. In contrast, luminal acidification in the cauda epididymidis is an Na+-dependent process. The specific apical Na+-dependent H+/base transport process(es) responsible for luminal acidification have not been identified. A potential clue as to the identity of these apical Na+-dependent H+/base transporter(s) is provided by similarities between the transport properties of the epididymis and the mammalian nephron. Specifically, the H+/base transport properties of caput epididymidis resemble the mammalian renal proximal tubule, whereas the distal epididymis and vas deferens have characteristics in common with renal collecting duct intercalated cells. Given the known expression of the Na+/H+ antiporter, NHE3, in the proximal tubule, and of the electroneutral sodium bicarbonate cotransporter, NBC3, in renal intercalated cells, we determined the localization of NHE3 and NBC3 in various regions of rat epididymis. NBC3 was highly expressed on the apical membrane of apical (narrow) cells in caput epididymidis, and light (clear) cells in corpus and cauda epididymidis. The number of cells expressing apical NBC3 was highest in cauda epididymidis. The localization of NBC3 in the epididymis was identical to the vacuolar H+-ATPase. The results indicate that colocalization of NBC3 and the vacuolar H+-ATPase is not restricted to kidney intercalated cells. Moreover, the close association of the two transporters appears to be a more generalized phenomenon in cells that express high levels of vacuolar H+-ATPase. Unlike NBC3, NHE3 was most highly expressed on the apical membrane of all epithelial cells in caput epididymidis, with less expression in the corpus, and no expression in the cauda. These results suggest that apical NBC3 and NHE3 potentially play an important role in mediating luminal H+/base transport in epididymis.