NHE3 mRNA Research Articles

Regulation of amino acid transporters in the rat remnant kidney

Partial renal ablation is associated with compensatory renal growth, significant azotaemia, a significant increase in fractional excretion of sodium and changes in solute transport. The present study evaluated the occurrence of adaptations in the remnant kidney, especially in renal amino acid transporters and sodium transporters and their putative role in sodium handling in the early stages (24 h and 1 week) after uninephrectomy. Wistar rats aged 8 weeks old were submitted to renal ablation of the right kidney--Unx rats (n = 10). 24 hours (n = 5) and 1 week (n = 5) after surgery, rats were anesthetized and the left kidney was removed. Urinary and plasmatic levels of catecholamines, sodium, urea and creatinine were measured. Gene expression of the amino acid and sodium transporters was determined by Real-time reverse transcription PCR. Protein expression was evaluated by Western blot using specific antibodies for the amino acid and sodium transporters. Uninephrectomized (Unx) rats for 24 h showed a lower urinary excretion of L-DOPA, dopamine and DOPAC than the corresponding Sham rats, accompanied by an increase in the expression of the Na(+)-K(+)-ATPase protein (64% increase). Unx rats for 1 week presented a hypertrophied remnant kidney, higher urine outflow and a approximately 2-fold increase in the fractional excretion of sodium. The NHE3 mRNA expression was significantly decreased in Unx rats throughout the study (approximately 20% decrease). LAT1 transcript and protein were consistently overexpressed at both 24 h and 1 week after uninephrectomy. In contrast, 4F2hc and LAT2 transcript abundance was lower in 24-h Unx rats than in Sham rats (a 36% decrease in both cases). These results provide evidence that the renal expression of the amino acid transporters LAT1, LAT2 and 4F2hc and the sodium transporters Na(+)-K(+)-ATPase and NHE3 is differently regulated following unilateral nephrectomy. In conclusion, this study allowed us to characterize the renal adaptations in the early stages after uninephrectomy, which showed a combined interaction of multiple mechanisms regulating sodium homeostasis including the renal dopaminergic system, and the abundance of amino acid transporters and sodium transporters.

Suppression of Na+/H+ exchanger isoform-3 in human inflammatory bowel disease: Lack of reversal by 5′-aminosalicylate treatment

Objective. Na+/H+ exchanger isoform 3 (NHE-3) is responsible for net uptake of NaCl and water from the gastrointestinal (GI) tract. However, its status in human inflammatory bowel diseases (IBDs) such as ulcerative colitis (UC) and Crohn's disease (CD) remains poorly understood. The aim of this study was to investigate the underlying mechanism of NHE-3 isoform expression and its modulation by 5′-aminosalicylate in human CD and UC. Material and methods. Subjects were divided into three groups: 1) controls; 2) untreated/new IBD cases (n=13) and 3) 5′- aminosalicylate-treated IBD patients (n_13). Subjects presenting with abdominal pain but with endoscopically normal colons served as normal controls. Inflammation was confirmed by the level of myeloperoxidase (MPO) activity, malondialdehyde (MDA) concentrations and by histologic evaluation. Expressions of NHE-3 protein and mRNA, sodium pump activity and IL-1β and TNF-α mRNA were estimated in the colonic biopsies using ECL-Western blot analysis, reverse transcription-polymerase chain reaction (RT-PCR) and enzyme assays. Results. The level of NHE-3 protein and sodium pump activity was reduced (p<0.05) in both the untreated and treated CD and UC patients. NHE-3 mRNA was reduced only in CD patients but not in those with UC. The treatment reversed the symptoms, but levels of MPO activity, MDA concentration, IL-1β, TNF-α and infiltration of inflammatory cells remained high with the exception of IL-1β mRNA in the treated patients. Conclusions. NHE-3 suppression is regulated differentially in CD and UC, which together with suppression of sodium pump activity will reduce NaCl and water uptake from the colonic lumen. These findings suggest a role of TNF-α in the regulation of NHE-3 expression in IBD.

Molecular cloning, tissue distribution and ontogenetic expression of sodium proton exchanger isoform 2 (NHE-2) mRNA in the small intestine of pigs

Molecular cloning, tissue distribution and ontogenetic regulation of sodium/proton exchanger isoform 2 (NHE-2) mRNA expression were evaluated in the pig small intestine during postnatal development. The 2872-bp porcine full cDNA sequence of the NHE-2 (EF672046) cloned in this study showed 80% and 70% homology with known human and mouse gene sequence, respectively. Hydrophobic prediction suggests 13 putative membrane-spanning domains within porcine NHE-2. The porcine NHE-2 mRNA was detected in the brain, liver, kidney, heart, lung, small intestine and muscle. The small intestine had the highest NHE-2 mRNA abundance and the brain, lung and liver had the lowest NHE-2 mRNA abundance (P < 0.05). Along the longitudinal axis, the duodenum had the highest NHE-2 mRNA abundance and the ileum and colon had the lowest NHE-2 mRNA abundance (P < 0.05). The NHE-2 mRNA level was increased from day 1 to day 26 in the duodenum (P < 0.05) and dropped dramatically on day 30 (P < 0.05). There is no difference between day 1 and day 7 (P > 0.05). After day 30, the NHE-2 mRNA level remained the same except on day 90 (P > 0.05). The mRNA expression of NHE-2 was not only differentially regulated by age but also differentially distributed along the small intestine of piglets at early stages and growing stages of life, which may contribute to changes in NHE activity.

Influence of curcumin on NHE-1 mRNA and hepatic stem cell proliferation and their correlations

Influence of curcumin on NHE-1 mRNA and hepatic stem cell proliferation and their correlations

Circadian expression of the Na+/H+ exchanger NHE3 in the mouse renal medulla

Renal tubular NHE3, the Na+/H+ exchanger, is a critical enzyme for electrolyte and acid-base homeostasis in the kidney. We previously demonstrated that the expression of this gene in the kidney followed a circadian rhythm directly regulated by clock genes acting on E-box elements present on its promoter region. In the present study, we further characterize the circadian expression of NHE3 in the mice kidney by in situ hybridization, and refine quantification of gene expression using real-time PCR combined with laser capture micro-dissection. We show NHE3 mRNA was strongly expressed in the inner stripe of the outer medulla and weakly in the cortex. Further realtime PCR data from dissected medullary nephron demonstrated clear circadian oscillations in the thick ascending limbs and the thin descending limbs, but not in the collecting ducts. The circadian changes of this molecule in the renal medulla may partially contribute to the circadian change of urinary electrolyte secretion.

Molecular and functional characterization of choline transporter in human colon carcinoma HT-29 cells

We examined the molecular and functional characterization of choline uptake in human colon carcinomas using the cell line HT-29. Furthermore, we explored the possible correlation between choline uptake and cell proliferation. Choline uptake was saturable and mediated by a single transport system. Interestingly, removal of Na + from the uptake buffer strongly enhanced choline uptake. This increase in component of choline uptake under Na +-free conditions was inhibited by a Na +/H + exchanger 1 (NHE1) inhibitor. Collapse of the plasma-membrane H + electrochemical gradient by a protonophore inhibited choline uptake. Choline uptake was inhibited by the choline analogue hemicholinium-3 (HC-3) and various organic cations, and was significantly decreased by acidification of the extracellular medium and by intracellular alkalinization. Real-time PCR revealed that choline transporter-like protein 1 (CTL1), CTL2, CTL4 and NHE1 mRNA are mainly expressed in HT-29 cells. Western blot and immunocytochemical analysis indicated that CTL1 protein was expressed in plasma membrane. The biochemical and pharmacological data indicated that CTL1 is functionally expressed in HT-29 cells and is responsible for choline uptake in these cells. We conclude that choline transporters, especially CTL1, use a directed H + gradient as a driving force, and its transport functions in co-operation with NHE1. Finally, cell proliferation was inhibited by HC-3 and tetrahexylammonium chloride (THA), which strongly inhibits choline uptake. Identification of this novel CTL1-mediated choline uptake system provides a potential new target for therapeutic intervention.

Tumor necrosis factor-{alpha} downregulates intestinal NHE8 expression by reducing basal promoter activity.

NHE8 transporter is a member of the sodium/hydrogen exchanger (NHE) family. This transporter protein is expressed at the apical membrane of epithelial cells of kidney and intestine and contributes to vectorial Na(+) transport in both tissues. Although NaCl absorption has been shown to be reduced in diarrhea associated with colitis and enteritis, little is known about the role of Na(+)/H(+) exchange and the involvement of NHE isoforms in the pathogenesis of inflammatory disorders and the mechanism of inflammation-associated diarrhea. This study investigated the role of NHE8 in the setting of inflammatory states. Jejunal mucosa was harvested from trinitrobenzene sulfonic acid (TNBS) colitis rats or lipopolysaccharide (LPS) rats for RNA extraction and brush-border membrane protein purification. The human NHE8 gene promoter was cloned from human genomic DNA and characterized in Caco-2 cells. The promoter was further used to study the mechanisms of TNF-alpha-mediated NHE8 expression downregulation in Caco-2 cells. Results from Western blot and real-time PCR indicated that NHE8 protein and mRNA were significantly reduced in TNBS rats and LPS rats. In Caco-2 cells, TNF-alpha produces similar reduction levels in the endogenous NHE8 mRNA expression observed in our in vivo studies. The downregulation of NHE8 expression mediated by TNF-alpha could be blocked by transcription inhibitor actinomycin D, suggesting the involvement of transcriptional regulation. Further studies indicated that the human NHE8 gene transcription could be activated by Sp3 transcriptional factor, and TNF-alpha inhibits human NHE8 expression by reducing Sp3 interaction at the minimal promoter region of the human NHE8 gene. In conclusion, our studies suggest that TNF-alpha decreases NHE8 expression in inflammation induced by TNBS and LPS, which may contribute to the diarrhea associated with inflammation.

Aldosterone regulation of intestinal Na absorption involves SGK-mediated changes in NHE3 and Na+pump activity

Aldosterone-induced intestinal Na(+) absorption is mediated by increased activities of apical membrane Na(+)/H(+) exchange (aNHE3) and basolateral membrane Na(+)-K(+)-ATPase (BLM-Na(+)-K(+)-ATPase) activities. Because the processes coordinating these events were not well understood, we investigated human intestinal Caco-2BBE cells where aldosterone increases within 2-4 h of aNHE3 and alpha-subunit of BLM-Na(+)-K(+)-ATPase, but not total abundance of these proteins. Although aldosterone activated Akt2 and serum glucorticoid kinase-1 (SGK-1), the latter through stimulation of phosphatidylinositol 3-kinase (PI3K), only the SGK-1 pathway mediated its effects on Na(+)-K(+)-ATPase. Ouabain inhibition of the early increase in aldosterone-induced Na(+)-K(+)-ATPase activation blocked most of the apical NHE3 insertion, possibly by inhibiting Na(+)-K(+)-ATPase-induced changes in intracellular sodium concentration ([Na](i)). Over the next 6-48 h, further increases in aNHE3 and BLM-Na(+)-K(+)-ATPase activity and total protein expression were observed to be largely mediated by aldosterone-activated SGK-1 pathway. Aldosterone-induced increases in NHE3 mRNA, for instance, could be inhibited by RNA silencing of SGK-1, but not Akt2. Additionally, aldosterone-induced increases in NHE3 promoter activity were blocked by silencing SGK-1 as well as pharmacological inhibition of PI3K. In conclusion, aldosterone-stimulated intestinal Na(+) absorption involves two phases. The first phase involves stimulation of PI3K, which increases SGK-dependent insertion and function of BLM-Na(+)-K(+)-ATPase and subsequent increased membrane insertion of aNHE3. The latter may be caused by Na(+)-K(+)-ATPase-induced changes in [Na] or transcellular Na flux. The second phase involves SGK-dependent increases in total NHE3 and Na(+)-K(+)-ATPase protein expression and activities. The coordination of apical and BLM transporters after aldosterone stimulation is therefore a complex process that requires multiple time- and interdependent cellular processes.

Defective jejunal and colonic salt absorption and alteredNa+/H+ exchanger 3 (NHE3) activity in NHE regulatory factor 1 (NHERF1) adaptor protein-deficient mice

We investigated the role of the Na(+)/H(+) exchanger regulatory factor 1 (NHERF1) on intestinal salt and water absorption, brush border membrane (BBM) morphology, and on the NHE3 mRNA expression, protein abundance, and transport activity in the murine intestine. NHERF1-deficient mice displayed reduced jejunal fluid absorption in vivo, as well as an attenuated in vitro Na(+) absorption in isolated jejunal and colonic, but not of ileal, mucosa. However, cAMP-mediated inhibition of both parameters remained intact. Acid-activated NHE3 transport rate was reduced in surface colonocytes, while its inhibition by cAMP and cGMP was normal. Immunodetection of NHE3 revealed normal NHE3 localization in the BBM of NHERF1 null mice, but NHE3 abundance, as measured by Western blot, was significantly reduced in isolated BBM from the small and large intestines. Furthermore, the microvilli in the proximal colon, but not in the small intestine, were significantly shorter in NHERF1 null mice. Additional knockout of PDZK1 (NHERF3), another member of the NHERF family of adaptor proteins, which binds to both NHE3 and NHERF1, further reduced basal NHE3 activity and caused complete loss of cAMP-mediated NHE3 inhibition. An activator of the exchange protein activated by cAMP (EPAC) had no effect on jejunal fluid absorption in vivo, but slightly inhibited NHE3 activity in surface colonocytes in vitro. In conclusion, NHERF1 has segment-specific effects on intestinal salt absorption, NHE3 transport rates, and NHE3 membrane abundance without affecting mRNA levels. However, unlike PDZK1, NHERF1 is not required for NHE3 regulation by cyclic nucleotides.

Involvement of Na +/H + exchanger 1 in advanced glycation end products-induced proliferation of vascular smooth muscle cell

In this present study, we examined the role of Na +/H + exchanger 1 (NHE1) in the cultured rat vascular smooth muscle cell (VSMC) proliferation induced by advanced glycation end products (AGEs). AGEs significantly increased the [ 3H] thymidine incorporation of VSMC. Cariporide, an NHE1 inhibitor, dose-dependently attenuated the AGEs-induced increase in cell DNA synthesis. Thus the effect of AGEs on NHE1 activity was next examined. The cariporide-dependent intracellular pH (pH i) was significantly increased after 24 h exposure to AGEs (10 μg/ml). The direct AGEs-induced NHE1 activation was measured by the Na +-dependent intracellular pH recovery from intracellular acidosis. AGEs can increase the NHE1 activity in a time- and concentration-dependent manner. Inhibition of either the receptor for AGEs (RAGE) by anti-RAGE or mitogen-activated protein kinases (MAPK) by PD98059 reversed the effect of AGEs on NHE1 activity. Reverse transcription (RT)-PCR analysis revealed that AGEs dose-dependently increased NHE1 mRNA at 24 h. These findings demonstrate NHE1 is required for in AGEs-induced proliferation of VSMC, and AGEs increase NHE1 activity via the MAPK pathway.

Na+/H+exchanger isoform 3 expressed in apical membrane of gill mitochondrion-rich cells in Mozambique tilapiaOreochromis mossambicus

A cDNA encoding a homolog of mammalian Na+/H+ exchanger isoform 3 (NHE3) has been cloned from gills of Mozambique tilapia Oreochromis mossambicus using reverse transcription-polymerase chain reaction (RT-PCR). The deduced amino acid sequence shared 48–56% homology with other vertebrate NHE3 homologs. RT-PCR revealed that tilapia NHE3 was expressed in the gill, kidney and intestine in tilapia adapted to fresh water (FW) and sea water (SW), suggesting its involvement in osmoregulation. We also examined NHE3 MRNA expression levels in the gills of FW and SW fish, using real-time quantitative PCR. NHE3 mRNA was highly expressed in the gills of FW fish, compared with those of SW fish. Whole-mount immunohistochemistry of the gill filaments revealed that tilapia NHE3 was localized in the apical region of mitochondrion-rich (MR) cells in FW-and SW-adapted tilapia. Abundance of NHE3 mRNA in FW gills and apical localization of NHE3 in gill MR cells suggest that NHE3 is involved in FW adaptation and acid-base regulation, in which Na+ is absorbed from external environments in exchange for H+ secretion.

Branchial expression and localization of SLC9A2 and SLC9A3 sodium/hydrogen exchangers and their possible role in acid–base regulation in freshwater rainbow trout (Oncorhynchus mykiss)

Experiments were conducted on adult rainbow trout (Oncorhynchus mykiss) to test the hypothesis that SLC9 Na+/H+ exchangers (SLC9A2, NHE2; and SLC9A3, NHE3) on the gill epithelium are localized specifically to a subset of mitochondria-rich cells (MRCs) that are unable to bind peanut lectin agglutinin (PNA). This cell type, termed the PNA- MRC, is a sub-type of MRC believed to function in Na+ uptake and acid excretion. A technique using biotinylated PNA was used to distinguish between the PNA- and PNA+ MRCs on fixed gill sections. In contrast to expectations, both NHE2 (mRNA) and NHE3 (protein) were confined to cells enriched with Na+/K+-ATPase and capable of binding PNA. Thus, in trout, NHE2 and NHE3 are localized to PNA+ MRCs, the cells previously believed to be responsible for Cl- uptake and base excretion. Levels of mRNA for NHE2, the predominant isoform in the gill, were increased during 72 h of hypercapnic acidosis; NHE3 mRNA and protein levels were unaffected. Because plasma cortisol levels were increased during hypercapnia (from 35.3+/-9.4 to 100.1+/-30.9 ng ml(-1)), the effects of experimentally elevated cortisol levels on NHE expression were investigated. The elevation of plasma cortisol using intraperitoneal implants caused a significant increase in NHE2 mRNA expression without affecting NHE3 mRNA or protein abundance. Thus, we suggest that NHE2 contributes to acid-base regulation during hypercapnia owing to its transcriptional regulation by cortisol. The finding of NHE expression in PNA+ MRCs is discussed with reference to current models of ionic and acid-base regulation in teleost fish.

Renal expression and localization of SLC9A3 sodium/hydrogen exchanger and its possible role in acid-base regulation in freshwater rainbow trout (Oncorhynchus mykiss)

Experiments were performed to assess the possible involvement of the Na(+)/H(+) exchanger isoform 3 (NHE3; SLC9A3) in renal acid-base regulation in adult rainbow trout (Oncorhynchus mykiss). NHE3 mRNA was expressed at high levels in the kidney relative to its paralog, NHE2. The results of in situ hybridization demonstrated an abundance of NHE3 mRNA in renal tubules. The combination of immunocytochemistry and histological staining revealed that NHE3 was confined to the apical membrane of proximal tubules, where it was colocalized with the vacuolar-type H(+)-ATPase. Levels of NHE3 protein (assessed by Western blotting) were increased during hypercapnia, likely as a result of increased transcription, as indicated by increasing levels of NHE3 mRNA (as determined by real-time PCR). Plasma cortisol concentration was increased during hypercapnia, and administration of exogenous cortisol caused a marked increase in NHE3 mRNA and protein. Thus we speculate that the elevation of plasma cortisol during hypercapnia contributes to transcriptional activation of NHE3 that ultimately promotes acid-base regulation by stimulating H(+) secretion and HCO(3)(-) reabsorption.

Astrocytic alkalinization by therapeutically relevant lithium concentrations: implications for myo-inositol depletion

One theory for therapeutic effects of the lithium ion (Li+) in bipolar disorder is that myo-inositol, needed for phospholipase C-mediated signaling, is depleted by Li(+)-induced inhibition of inositolphosphate hydrolysis or of myo-inositol uptake, an effect demonstrated in cultured mouse astrocytes at high myo-inositol concentrations. In contrast, myo-inositol uptake is inhibited at low concentrations, reflecting that it occurs both by the high-affinity Na(+)-dependent myo-inositol transporter (SMIT) and the lower-affinity H(+)-dependent inositol transporter (HMIT). Increased intracellular pH (pHi) stimulates SMIT but inhibits HMIT, suggesting that the effect of Li+ could be caused by intracellular alkalinization. In this study, we therefore investigated Li+ effects on intracellular pH in astrocytes, measured by 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) fluorescence. Chronic treatment with the therapeutically relevant Li+ concentration of 1 mM for 2 or 3 weeks increased pHi by approximately 0.10, whereas 0.5 mM was ineffective, and 2 mM caused a larger increase. The alkalinization resulted from acute stimulation of the Na+/H+ exchanger (NHE) by extracellular Li+, demonstrated after acid load with NH4Cl. In response to continuous stimulation, NHE1 mRNA was down-regulated, but protein was not. Chronic treatment with pharmacologically relevant Li+ concentrations increases pHi in astrocytes, creating conditions for decreased uptake of high myo-inositol concentrations and increased uptake of low concentrations. The pharmacological relevance of this effect is supported by literature data suggesting brain acidosis in bipolar patients and by preliminary observations that carbamazepine and valproate also increase pHi in astrocytes. Stimulation of NHE1-stimulated sodium ion uptake might also trigger uptake of chloride ions and osmotically obliged water.

The COOH terminus of megalin regulates gene expression in opossum kidney proximal tubule cells

We recently reported that megalin is subjected to regulated intramembrane proteolysis (RIP) and includes 1) protein kinase C (PKC)-regulated, metalloprotease-mediated ectodomain shedding producing a membrane-bound megalin COOH-terminal fragment (MCTF) and 2) gamma-secretase-mediated cleavage of the MCTF producing a soluble megalin intracellular domain (MICD). Based on studies of RIP of other receptors, the MICD is predicted to target to the nucleus and regulate gene expression. To determine whether RIP of megalin regulates proximal tubule gene expression, we stably expressed the transfected MCTF (tMCTF) or transfected MICD (tMICD) in opossum kidney proximal tubule (OKP) cells and examined the resulting phenotype. Immunoblotting and immunocytochemical analysis of tMCTF cells showed the tMCTF was expressed and constitutively processed by gamma-secretase. Analysis of specific protein expression in tMCTF- and tMICD-transfected cells using Western blot showed endogenous megalin and Na(+)/H(+) exchanger 3 (NHE3) protein expression to be dramatically lower than that of control cells. Expression of other proteins including myosin VI, beta-adaptin, and the Na-K-ATPase appeared unchanged. Analysis of specific mRNA expression using quantitative real-time PCR showed megalin and NHE3 mRNA levels were significantly lower in tMCTF- and tMICD-transfected cells compared with controls. Inhibition of gamma-secretase activity in tMCTF cells resulted in an 8- to 10-fold recovery of megalin mRNA within 4 h. These data show that the COOH-terminal domain of megalin regulates expression of specific proteins in OKP cells and provides the first evidence that RIP of megalin may be part of a signaling pathway linking protein absorption and gene expression in proximal tubule.

M1721 Role of Organic Cation Transporters (Oct1, Oct2 and Oct3) in the Uptake of the Duodenal Ulcerogen Cysteamine in Rat Duodenal Mucosa and in Rat Small Intestinal Epithelial Cell Line IEC-6: Use of siRNA Technology

SGLT1 siRNA treated cells, n=3). Kinetic studies demonstrated that the mechanism of SGLT1 siRNA mediated stimulation of Na:H exchange in IEC-18 cells was secondary to a significant increase in the Vmax (10.0 ±1.1 nmol/mg protein/30 sec in control cells and 31.0 ± 2.5 in SGLT1 siRNA treated cells, n=3, p<0.05) without an alteration in the affinity of the exchanger for Na. RTQ-PCR demonstrated significant decrease in SGLT1 mRNA while NHE3 mRNA was significantly increased with SGLT1 siRNA treatment of IEC-18 cells. Western blot studies demonstrated that whereas SGLT1 protein was nearly absent NHE3 protein increased 2 fold SGLT1 siRNA treated IEC 18 cells. Conclusions: A reduction in BBM SGLT1 protein levels directly stimulates BBMNHE3 in IEC-18 cells. Themechanism of this stimulation is secondary to an increase in NHE3 transporter numbers. Thus, these data indicate that BBM SGLT compensatorily regulates BBM NHE3 in intestinal epithelial cells to maintain intracellular Na homeostasis.

The role of SGK-1 in angiotensin II-mediated sodium reabsorption in human proximal tubular cells

The role of angiotensin II (Ang II) in mediating excessive sodium reabsorption in diabetic nephropathy is recognized. Serine-glucocorticoid kinase-1 (SGK-1) increases sodium-hydrogen exchanger-3 (NHE3) expression and is known to be upregulated in in vitro and in vivo models of diabetic nephropathy. However, a link between Ang II and SGK-1 in diabetic nephropathy has not been established. Ang II production in cultured human proximal tubular cells was measured under normal (5 mM) and high (25 mM) glucose conditions. The Ang II type 1 receptor was identified by RT-PCR. SGK-1 and NHE3 mRNA and protein expression was measured in proximal tubule cells (PTCs) exposed to Ang II. EIPA inhibitable changes in cell sodium uptake were undertaken to confirm that alterations in NHE3 mRNA and protein were reflected in transport activity. SGK-1 was silenced in the PTCs using small interfering RNA to determine the role of SGK-1 in mediating Ang II-induced increases in NHE3-mediated sodium uptake. Ang II production by PTCs was significantly increased by exposure to high glucose (P < 0.02). Ang II increased NHE3 and SGK-1 mRNA expression to 275 +/- 30% (P < 0.02) and 130 +/- 10% (P < 0.05) respectively. Silencing of SGK-1 reduced Ang II-stimulated NHE3 protein expression to 49.8 +/- 6.1% (P < 0.05) of control levels. SGK-1 silencing abolished increases in (22)Na(+) uptake seen in Ang II-treated cells to 86.7 +/- 1.6% of control values. These data suggest that increased sodium reabsorption in renal proximal tubular cells considered to be due to Ang II in diabetes mellitus is mediated through SGK-1 expression.

Mechanisms underlying the long-term regulation of NHE3 by parathyroid hormone

The activity of the Na(+)/H(+) exchanger NHE3 is regulated by a number of factors including parathyroid hormone (PTH). In the current study, we used a renal epithelial cell line, the opossum kidney (OKP) cell, to elucidate the mechanisms underlying the long-term effects of PTH on NHE3 transport activity and expression. We observed that NHE3 activity was reduced 6 h after addition of PTH, and this reduction persisted almost unaltered after 24 h. The decrease in activity was associated with diminished NHE3 cell surface expression at 6, 16, and 24 h after PTH addition, total cellular NHE3 protein at 16 and 24 h, and NHE3 mRNA abundance at 24 h. The lower levels of NHE3 mRNA were associated to a small, but significant, decrease in mRNA stability. Additionally, by analyzing the rat NHE3 gene promoter activity in OKP cells, we verified that the regulatory region spanning the segment -152 to +55 was mildly reduced under the influence of PTH. This effect was completely abolished by the presence of the PKA inhibitor KT 5720. In conclusion, long-term exposure to PTH results in reduction of NHE3 mRNA levels due to a PKA-dependent inhibitory effect on the NHE3 promoter and a small reduction of mRNA half-life, and decrease in the total amount of protein which is preceded by endocytosis of the apical surface NHE3. The decreased NHE3 expression is likely to be responsible for the reduction of sodium, bicarbonate, and fluid reabsorption in the proximal tubule consistently perceived in experimental models of PTH disorders.

Molecular Cloning, Tissue Distribution and Ontogenetic Expression of Sodium Proton Exchanger (NHE2) mRNA in the Small Intestine of Pigs

Molecular cloning, tissue distribution and ontogenetic regulation of sodium/proton exchanger isoform 2 (NHE2) mRNA expression were evaluated in the pig small intestine during postnatal development. The 2872‐bp porcine full cDNA sequence of the NHE2 (EF672046) cloned in this study showed 80% and 70% homology with known human and mouse gene sequence, respectively. Hydrophobic prediction suggests 13 putative membrane‐spanning domains within porcine NHE2. The porcine NHE2 mRNA was detected in brain, liver, kidney, heart, lung, small intestine and muscle. The small intestine had the highest NHE2 mRNA abundance and the brain, lung and liver had the lowest NHE2 mRNA abundance (P &lt; 0.05). Along the longitudinal axis, the duodenum had the highest NHE2 mRNA abundance and the ileum and colon had the lowest NHE2 mRNA abundance (P &lt; 0.05). The NHE2 mRNA level was increased from the day 1 to day 26 in the duodenum (P &lt; 0.05) and dropped dramatically on day 30 (P &lt; 0.05). There is no difference between day 1 and day 7 (P &gt; 0.05). After day 30, the NHE2 mRNA level stayed at the same except on day 90 (P &gt; 0.05). The mRNA expression of NHE2 was not only differentially regulated by age but also differentially distributed along the small intestine of piglets at early stages and growing stages of life, which may contribute to changes in NHE activity.

Molecular and immunological characterization of Na+/H+ antiporter (NHE3) in the gills of a marine teleost (Myoxocephalus octodecemspinosus)

The marine long horn sculpin compensates for metabolic acidosis by excretion of net acid equivalents across the branchial epithelium. Transfer of H+ is dependent on external Na+ and can be inhibited by amiloride. We have evidence that at least four orthologs of mammalian Na+/H+ exchangers are expressed in the gills (NHE1–3, 8). In this study, we have cloned a full open reading frame for gill NHE3. mRNA (Northerns; 4.3–4.8 kb) and protein expression (Westerns; ~104 kd) band sizes were similar to mammalian NHE3. In situ hybridization with anti‐sense probe recognized cells at the base of the lamellae and in the interlamellar space. This location was confirmed with florescent immunohistochemistry using species specific antibodies. Strong apical NHE3 signal was detected in gill cells within the interlamellar region. Na+/K+‐ATPase was colocalized to these cells as well. Sculpin specific antibodies against H+‐ATPase detected cytoplasmic expression of this protein in a separate subpopulation of gill cells. Paralleling in vivo net H+ efflux, NHE3 mRNA signal increased two hours after an infused acid load while apical NHE3 protein expression was detected in both control and acid loaded animals. Thus it appears that an NHE3 ortholog is coexpressed in Na+/K+‐ATPase rich gill cells and likely contributes to H+ excretion for acid‐base regulation. Supported by NSF‐IBN‐0616187 to JBC.