Transcriptional regulation of the sodium-sulfate cotransporter NaSi-1 gene

Inorganic sulfate is one of the most abundant anions in mammalian plasma and is essential for proper cell growth and development, as well as detoxification and activation of many biological compounds. To date, little is understood how physiological levels of sulfate are maintained in the body. Our studies, and of others, have identified the NAS(i)-1 protein to be a functional sulfate transporter in the kidney and intestine, and due to this localization, constitutes a strong candidate gene for maintaining body sulfate homeostasis. Several factors, including hormones and metabolic conditions, have been shown to alter NAS(i)-1 mRNA and protein levels in vivo. In this study, we describe the transcriptional regulation of NaSi-1, with a focus on the mouse NaSi-1 gene (Nas1) that was recently cloned in our laboratory. Vitamin D (1,25-(OH)(2)D-3) and thyroid hormone (T-3) led to an increase in Nas1 promoter activity in OK cells. Mutational analysis of the Nas1 promoter resulted in identification of a direct repeat 6-type vitamin-D-responsive element (DR6 VDRE) at -525 to -508 and an imperfect inverted repeat 0-type T-3 responsive element (IRO T3RE) at -426 to -425 which conferred 1,25-(OH)(2)D-3 and T-3 responsiveness respectively. These findings suggest for vitamin D and thyroid hormone regulation of NaSi-1, may provide important clues to the physiological control of sulfate homeostasis.

WNK3 abrogates the NEDD4-2-mediated inhibition of the renal Na+-Cl- cotransporter.

The serine/threonine kinase WNK3 and the ubiquitin-protein ligase NEDD4-2 are key regulators of the thiazide-sensitive Na+-Cl- cotransporter (NCC), WNK3 as an activator and NEDD2-4 as an inhibitor. Nedd4-2 was identified as an interacting partner of WNK3 through a glutathione-S-transferase pull-down assay using the N-terminal domain of WNK3, combined with LC-MS/MS analysis. This was validated by coimmunoprecipitation of WNK3 and NEDD4-2 expressed in HEK293 cells. Our data also revealed that the interaction between Nedd4-2 and WNK3 does not involve the PY-like motif found in WNK3. The level of WNK3 ubiquitylation did not change when NEDD4-2 was expressed in HEK293 cells. Moreover, in contrast to SGK1, WNK3 did not phosphorylate NEDD4-2 on S222 or S328. Coimmunoprecipitation assays showed that WNK3 does not regulate the interaction between NCC and NEDD4-2. Interestingly, in Xenopus laevis oocytes, WNK3 was able to recover the SGK1-resistant NEDD4-2 S222A/S328A-mediated inhibition of NCC and further activate NCC. Furthermore, elimination of the SPAK binding site in the kinase domain of WNK3 (WNK3-F242A, which lacks the capacity to bind the serine/threonine kinase SPAK) prevented the WNK3 NCC-activating effect, but not the Nedd4-2-inhibitory effect. Together, these results suggest that a novel role for WNK3 on NCC expression at the plasma membrane, an effect apparently independent of the SPAK kinase and the aldosterone-SGK1 pathway.

Regulation of the NaCI cotransporter by the SGK1-Nedd4-2 pathway

SummaryRegulation of renal Na+ transport is essential for controlling blood pressure, as well as Na+ and K+ homeostasis. Aldosterone stimulates Na+ reabsorption in the aldosterone-sensitive distal nephron (ASDN), via the Na+-CI" cotransporter (NCC) in the distal convoluted tubule (DCT), and the epithelial Na+ channel (ENaC) in the late DCT, connecting tubule and collecting duct. Importantly, aldosterone increases NCC protein expression by an unknown post-translational mechanism. The ubiquitin-protein ligase Nedd4-2 is expressed along the ASDN and regulates ENaC: under aldosterone induction, the serum/glucocorticoid-regulated kinase SGK1 phosphorylates Nedd4-2 on S328, thus preventing the Nedd4-2/ENaC interaction, ubiquitylation and degradation of the channel. Here, we present evidence that Nedd4-2 regulates NCC. In transfected HEK293 cells, Nedd4-2 co-immunoprecipitates with NCC and stimulates NCC ubiquitylation at the cell surface. In Xenopus laevis oocytes, co- expression of NCC with wild-type Nedd4-2, but not its catalytically inactive mutant, strongly decreases NCC activity and surface expression. This inhibition is prevented by SGK1 in a kinase-dependent manner. Moreover, we show that NCC expression is up-regulated in inducible renal tubule-specific Nedd4-2 knockout mice and in mDCT15 cells silenced for Nedd4-2. On the other hand, in inducible renal tubule-specific SGK1 knockout mice, NCC expression is down-regulated.Interestingly, in contrast to ENaC, Nedd4-2-mediated NCC inhibition is independent of a PY motif in NCC. Moreover, whereas single mutations of Nedd4-2 S328 or S222 to alanine do not interfere with SGK1 action, the double mutation enhances Nedd4-2 activity and abolishes SGK1-dependent inhibition. These results indicate that NCC expression and activity is controlled by a regulatory pathway involving SGK1 and Nedd4-2, and provides an explanation for the well-known aldosterone-induced increase in NCC protein expression.RésuméLa régulation du transport de sodium est cruciale dans le maintien de la pression artérielle. L'aldostérone stimule la réabsorption de Na+ dans la partie du néphron sensible à l'aldostérone (ASDN), via le co-transporteur Na+-CI" (NCC) au niveau du tubule contourné distale et via le canal à sodium (Epithelial Na+ Channel ; ENaC) dans la deuxième partie du tubule contourné distale, dans le tube connecteur et le tube collecteur. L'aldostérone augmente l'expression de NCC au niveau protéique par un mécanisme non élucidé. La protéine ubiquitine ligase Nedd4-2 est exprimée tout le long du néphron sensible à l'aldostérone. ENaC est connu pour être régulé par Nedd4-2. Suite à une stimulation par l'aldostérone, la kinase Ser/Thr SGK1 phosphoryle Nedd4-2, ce qui empêche l'interaction entre Nedd4-2 et ENaC. Dans des cellules HEK293 transfectées, nous avons montré que Nedd4-2 interagit avec le co-transporteur NCC et stimule l'ubiquitylation de NCC à la surface. Nous avons montré dans les oocytes de Xenopus laevis que l'expression de NCC avec Nedd4-2 diminue l'activité du co-transporteur. Cette diminution n'est pas observée lorsqu'on exprime NCC avec le mutant inactif de Nedd4-2. Cette inhibition de NCC est contrée par SGK1. L'effet de SGK1 sur NCC dépend de son activité kinase. Nous avons montré dans des souris knock-out pour Nedd4-2, dans le néphron et de manière inductible, que l'expression de NCC est augmentée. Nous avons également montré que la suppression de la protéine Nedd4-2 dans les cellules mDCT15 provoque l'augmentation de NCC. Au contraire dans les souris knock-out pour la kinase SGK1, dans le néphron et de manière inductible, nous observons une diminution de la protéine NCC. Contrairement à ce qui a été montré pour le canal ENaC l'inhibition de NCC par Nedd4-2 est indépendante des motifs PY. De plus, La mutation des sérines 328 ou 222 sur Nedd4-2 en alanine n'interfère pas avec l'action de SGK1 pour prévenir l'inhibition. Par contre, la double mutation, les sérines 222 et 328 mutées en alanine, augmente l'action de Nedd4-2 sur l'activité de NCC et prévient l'effet de SGK1. Ces résultats montrent que l'expression et l'activité de NCC sont contrôlées par une voie de régulation impliquant Nedd4-2-SGK1 et nous fournissent une explication pour l'augmentation de NCC observé après une induction avec l'aldostérone.Résumé large publicOn estime que des millions de personnes seraient hypertendues. L'hypertension artérielle est responsable d'environ 8 millions de décès par ans dans le monde. L'hypertension est responsable de la moitié environs des accidents cardiaques, mais aussi des accidents vasculaires cérébraux. Il est très important de comprendre les mécanismes qui se trouvent derrière cette pathologie.Le co-transporteur NCC joue un grand rôle dans le maintien de la balance sodique. Il a été montré que des perturbations dans l'expression de NCC pouvaient engendrer de l'hypertension.Le co-transporteur NCC est exprimé dans la partie distale du néphron, l'unité fonctionnelle du rein. Plusieurs études ont montrées que NCC était sous le contrôle de l'hormone aldostérone.Le travail de cette thèse consiste à étudier les mécanismes impliqués dans la régulation de NCC. On a ainsi pu montrer que NCC interagit avec la protéine ubiquitine ligase Nedd4-2. La protéine Nedd4-2 diminue l'expression de NCC à la surface cellulaire et aussi son activité Nous avons également montré que la kinase SGK1 pouvait prévenir l'interaction entre Nedd4-2 et NCC par phosphorylation de Nedd4-2. Nous avons montré dans des souris deletée pour Nedd4-2, dans le néphron, que l'expression de NCC est augmentée. Nous avons également montré que la suppression de la protéine Nedd4-2 dans les cellules mDCT15 provoque l'augmentation de NCC. Au contraire, dans les souris deletée pour la kinase SGK1, dans le néphron, nous observons une diminution de la protéine NCC. La connaissance des processus impliqués dans la régulation du co-transporteur NCC pourrait amener au développement de nouveau médicaments pour soigner l'hypertension.

Nedd4-2 modulates renal Na+-Cl- cotransporter via the aldosterone-SGK1-Nedd4-2 pathway.

Regulation of renal Na(+) transport is essential for controlling blood pressure, as well as Na(+) and K(+) homeostasis. Aldosterone stimulates Na(+) reabsorption by the Na(+)-Cl(-) cotransporter (NCC) in the distal convoluted tubule (DCT) and by the epithelial Na(+) channel (ENaC) in the late DCT, connecting tubule, and collecting duct. Aldosterone increases ENaC expression by inhibiting the channel's ubiquitylation and degradation; aldosterone promotes serum-glucocorticoid-regulated kinase SGK1-mediated phosphorylation of the ubiquitin-protein ligase Nedd4-2 on serine 328, which prevents the Nedd4-2/ENaC interaction. It is important to note that aldosterone increases NCC protein expression by an unknown post-translational mechanism. Here, we present evidence that Nedd4-2 coimmunoprecipitated with NCC and stimulated NCC ubiquitylation at the surface of transfected HEK293 cells. In Xenopus laevis oocytes, coexpression of NCC with wild-type Nedd4-2, but not its catalytically inactive mutant, strongly decreased NCC activity and surface expression. SGK1 prevented this inhibition in a kinase-dependent manner. Furthermore, deficiency of Nedd4-2 in the renal tubules of mice and in cultured mDCT(15) cells upregulated NCC. In contrast to ENaC, Nedd4-2-mediated inhibition of NCC did not require the PY-like motif of NCC. Moreover, the mutation of Nedd4-2 at either serine 328 or 222 did not affect SGK1 action, and mutation at both sites enhanced Nedd4-2 activity and abolished SGK1-dependent inhibition. Taken together, these results suggest that aldosterone modulates NCC protein expression via a pathway involving SGK1 and Nedd4-2 and provides an explanation for the well-known aldosterone-induced increase in NCC protein expression.

Nedd4-2 modulates renal Na+-Cl- cotransporter via the aldosterone-SGK1-Nedd4-2 pathway.

Regulation of renal Na(+) transport is essential for controlling blood pressure, as well as Na(+) and K(+) homeostasis. Aldosterone stimulates Na(+) reabsorption by the Na(+)-Cl(-) cotransporter (NCC) in the distal convoluted tubule (DCT) and by the epithelial Na(+) channel (ENaC) in the late DCT, connecting tubule, and collecting duct. Aldosterone increases ENaC expression by inhibiting the channel's ubiquitylation and degradation; aldosterone promotes serum-glucocorticoid-regulated kinase SGK1-mediated phosphorylation of the ubiquitin-protein ligase Nedd4-2 on serine 328, which prevents the Nedd4-2/ENaC interaction. It is important to note that aldosterone increases NCC protein expression by an unknown post-translational mechanism. Here, we present evidence that Nedd4-2 coimmunoprecipitated with NCC and stimulated NCC ubiquitylation at the surface of transfected HEK293 cells. In Xenopus laevis oocytes, coexpression of NCC with wild-type Nedd4-2, but not its catalytically inactive mutant, strongly decreased NCC activity and surface expression. SGK1 prevented this inhibition in a kinase-dependent manner. Furthermore, deficiency of Nedd4-2 in the renal tubules of mice and in cultured mDCT(15) cells upregulated NCC. In contrast to ENaC, Nedd4-2-mediated inhibition of NCC did not require the PY-like motif of NCC. Moreover, the mutation of Nedd4-2 at either serine 328 or 222 did not affect SGK1 action, and mutation at both sites enhanced Nedd4-2 activity and abolished SGK1-dependent inhibition. Taken together, these results suggest that aldosterone modulates NCC protein expression via a pathway involving SGK1 and Nedd4-2 and provides an explanation for the well-known aldosterone-induced increase in NCC protein expression.

An Ion Transport-Independent Role for the Cation-Chloride Cotransporter KCC2 in Dendritic Spinogenesis In Vivo.

The neuron-specific K-Cl cotransporter, KCC2, is highly expressed in the vicinity of excitatory synapses in pyramidal neurons, and recent in vitro data suggest that this protein plays a role in the development of dendritic spines. The in vivo relevance of these observations is, however, unknown. Using in utero electroporation combined with post hoc iontophoretic injection of Lucifer Yellow, we show that premature expression of KCC2 induces a highly significant and permanent increase in dendritic spine density of layer 2/3 pyramidal neurons in the somatosensory cortex. Whole-cell recordings revealed that this increased spine density is correlated with an enhanced spontaneous excitatory activity in KCC2-transfected neurons. Precocious expression of the N-terminal deleted form of KCC2, which lacks the chloride transporter function, also increased spine density. In contrast, no effect on spine density was observed following in utero electroporation of a point mutant of KCC2 (KCC2-C568A) where both the cotransporter function and the interaction with the cytoskeleton are disrupted. Transfection of the C-terminal domain of KCC2, a region involved in the interaction with the dendritic cytoskeleton, also increased spine density. Collectively, these results demonstrate a role for KCC2 in excitatory synaptogenesis in vivo through a mechanism that is independent of its ion transport function.

Na plus -Glucose Cotransporter SGLT1 Protein in Salivary Glands: Potential Involvement in the Diabetes-Induced Decrease in Salivary Flow

Oral health complications in diabetes include decreased salivary secretion. The SLC5A1 gene encodes the Na(+)-glucose cotransporter SGLT1 protein, which not only transports glucose, but also acts as a water channel. Since SLC5A1 expression is altered in kidneys of diabetic subjects, we hypothesize that it could also be altered in salivary glands, contributing to diabetic dysfunction. The present study shows a diabetes-induced decrease (p < 0.001) in salivary secretion, which was accompanied by enhanced (p < 0.05) SGLT1 mRNA expression in parotid (50%) and submandibular (30%) glands. Immunohistochemical analysis of parotid gland of diabetic rats revealed that SGLT1 protein expression increased in the luminal membrane of ductal cells, which can stimulate water reabsorption from primary saliva. Furthermore, SGLT1 protein was reduced in myoepithelial cells of the parotid from diabetic animals, and that, by reducing cellular contractile activity, might also be related to reduced salivary flux. Six-day insulin-treated diabetic rats reversed all alterations. In conclusion, diabetes increases SLC5A1 gene expression in salivary glands, increasing the SGLT1 protein content in the luminal membrane of ductal cells, which, by increasing water reabsorption, might explain the diabetes-induced decrease in salivary secretion.

Synthesis, maturation, and trafficking of human Na+-dicarboxylate cotransporter NaDC1 requires the chaperone activity of cyclophilin B

Renal excretion of citrate, an inhibitor of calcium stone formation, is controlled mainly by reabsorption via the apical Na(+)-dicarboxylate cotransporter NaDC1 (SLC13A2) in the proximal tubule. Recently, it has been shown that the protein phosphatase calcineurin inhibitors cyclosporin A (CsA) and FK-506 induce hypocitraturia, a risk factor for nephrolithiasis in kidney transplant patients, but apparently through urine acidification. This suggests that these agents up-regulate NaDC1 activity. Using the Xenopus lævis oocyte and HEK293 cell expression systems, we examined first the effect of both anti-calcineurins on NaDC1 activity and expression. While FK-506 had no effect, CsA reduced NaDC1-mediated citrate transport by lowering heterologous carrier expression (as well as endogenous carrier expression in HEK293 cells), indicating that calcineurin is not involved. Given that CsA also binds specifically to cyclophilins, we determined next whether such proteins could account for the observed changes by examining the effect of selected cyclophilin wild types and mutants on NaDC1 activity and cyclophilin-specific siRNA. Interestingly, our data show that the cyclophilin isoform B is likely responsible for down-regulation of carrier expression by CsA and that it does so via its chaperone activity on NaDC1 (by direct interaction) rather than its rotamase activity. We have thus identified for the first time a regulatory partner for NaDC1, and have gained novel mechanistic insight into the effect of CsA on renal citrate transport and kidney stone disease, as well as into the regulation of membrane transporters in general.

Phosphoregulation of K(+)-Cl(-) cotransporter 4 during changes in intracellular Cl(-) and cell volume

It has long been stated that the K(+)-Cl(-) cotransporters (KCCs) are activated during cell swelling through dephosphorylation of their cytoplasmic domains by a protein phosphatase (PP) but that other enzymes are involved by targeting this PP or the KCCs directly. To date, however, the role of signaling intermediates in KCC regulation has been deduced from indirect evidence rather than in vitro phosphorylation studies, and examined after simulation of ion transport through cell swelling or N-ethylmaleimide treatment. In this study, the oocyte expression system was used to examine the effects of changes in cell volume (C(VOL)) and intracellular [Cl(-)] ([Cl(-)](i)) on the activity and phosphorylation levels (P(LEV)) of KCC4, and determine whether these effects are mediated by PP1 or phorbol myristate acetate (PMA)-sensitive effectors. We found that (1) low [Cl(-)](i) or low C(VOL) leads to decreased activity but increased P(LEV), (2) high C(VOL) leads to increased activity but no decrease in P(LEV) and (3) calyculin A (Cal A) or PMA treatment leads to decreased activity but no increase in P(LEV). Thus, we have shown for the first time that one of the KCCs can be regulated through direct phosphorylation, that changes in [Cl(-)](i) or C(VOL) modify the activity of signaling enzymes at carrier sites, and that the effectors directly involved do not include a Cal A-sensitive PP in contrast to the widely held view. J. Cell. Physiol. 219: 787-796, 2009. (c) 2009 Wiley-Liss, Inc.

Regulation of sodium/glucose cotransporter expression in LLC-PK$\sb1$ cells

Expression of the Na$\sp+$/glucose cotransporter (SGLT1), a differentiated function of the pig kidney epithelial cell line LLC-PK$\sb1$ derived from proximal tubule, was further investigated. The differentiation inducer hexamethylene bisacetamide (HMBA) and IBMX, an inhibitor of cAMP phosphodiesterase, each stimulated a significant increase in Na$\sp+$/glucose cotransport activity, levels of the 75 kD cotransporter subunit and steady-state levels of the SGLT1 message. The action of HMBA is associated with involvement of polyamines and protein kinase C, and is synergistic with cAMP. We provide evidence that cAMP-elevating agents increase Na$\sp+$/glucose cotransporter expression, at least in part, via a post-transcriptional mechanism. Two molecular species of SGLT1 mRNA (3.9 kb and 2.2 kb) are transcribed from the same gene in LLC-PK$\sb1$ cells and differ only in the length of the 3$\sp\prime$ untranslated region (3$\sp\prime$ UTR). cAMP elevation differentially stabilized the 3.9 kb SGLT1 transcript from degradation but not the 22 kb species. UV-cross-linking and label transfer experiments indicated that cyclic AMP elevation was associated with formation of a 48 kD protein complex with a specific domain within the 3$\sp\prime$ UTR of SGLT1 mRNA. The binding was competitively inhibited by poly (U) and other U-rich RNA species such as c-fos ARE, and modulated by a protein kinase A-mediated phosphorylation/dephosphorylation mechanism. The binding site was mapped to a 120-nucleotide 3$\sp\prime$ UTR sequence which contains a uridine-rich region (URE). Our study provides the first demonstration that renal SGLT1 is post-transcriptionally regulated by a phosphorylation/dephosphorylation mechanism, and provides a deeper insight into gene regulation of this physiologically important cotransporter. ^

A sea urchin Na+K+2Cl- cotransporter is involved in the maintenance of calcification-relevant cytoplasmic cords in Strongylocentrotus droebachiensis larvae

The cellular mechanisms of calcification in sea urchin larvae are still not well understood. Primary mesenchyme cells within the larval body cavity form a syncytium to secrete CaCO3 spicules from intracellular amorphous CaCO3 (ACC) stores. We studied the role of Na+K+2Cl- cotransporter (NKCC) in intracellular ACC accumulation and larval spicule formation of Strongylocentrotus droebachiensis. First, we incubated growing larvae with three different loop diuretics (azosemide, bumetanide, and furosemide) and established concentration-response curves. All loop diuretics were able to inhibit calcification already at concentrations that specifically inhibit NKCC. Calcification was most effectively inhibited by azosemide (IC50 = 6.5 µM), while larval mortality and swimming ability were not negatively impacted by the treatment. The inhibition by bumetanide (IC50 = 26.4 µM) and furosemide (IC50 = 315.4 µM) resembled the pharmacological fingerprint of the mammalian NKCC1 isoform. We further examined the effect of azosemide on the maintenance of cytoplasmic cords and on the occurrence of calcification vesicles using fluorescent dyes (calcein, FM1-43). Fifty micromolars of azosemide inhibited the maintenance of cytoplasmic cords and resulted in increased calcein fluorescence within calcification vesicles. The expression of NKCC in S. droebachiensis was verified by PCR and Western blot with a specific NKCC antibody. In summary, the pharmacological profile of loop diuretics and their specific effects on calcification in sea urchin larvae suggest that they act by inhibition of NKCC via repression of cytoplasmic cord formation and maintenance.

The role of transmembrane domain 2 in cation transport by the Na–K–Cl cotransporter

The human and shark Na–K–Cl cotransporters (NKCC), although 74% identical in amino acid sequence, exhibit marked differences in ion transport and bumetanide binding. We have utilized shark–human chimeras of NKCC1 to search for regions that confer the kinetic differences. Two chimeras (hs3.1 and its reverse sh3.1) with a junction point located at the beginning of the third transmembrane domain were examined after stable transfection in HEK-293 cells. Each carried out bumetanide-sensitive 86Rb influx with cation affinities intermediate between shark and human cotransporters. In conjunction with the previous finding that the N and C termini are not responsible for differences in ion transport, the current observations identify the second transmembrane domain as playing an important role. Site-specific mutagenesis of two pairs of residues in this domain revealed that one pair is indeed involved in the difference in Na affinity, and a second pair is involved in the difference in Rb affinity. Substitution of the same residues with corresponding residues from NKCC2 or the Na-Cl cotransporter resulted in cation affinity changes, consistent with the hypothesis that alternative splicing of transmembrane domain 2 endows different versions of NKCC2 with unique kinetic behaviors. None of the changes in transmembrane domain 2 was found to substantially affect Km(Cl), demonstrating that the affinity difference for Cl is specified by the region beyond predicted transmembrane domain 3. Finally, unlike Cl, bumetanide binding was strongly affected by shark–human replacement of transmembrane domain 2, indicating that the bumetanide-binding site is not the same as the Cl-binding site.

Cotransport of water by the Na+/glucose cotransporter

Water is transported across epithelial membranes in the absence of any hydrostatic or osmotic gradients. A prime example is the small intestine, where 10 liters of water are absorbed each day. Although water absorption is secondary to active solute transport, the coupling mechanism between solute and water flow is not understood. We have tested the hypothesis that water transport is directly linked to solute transport by cotransport proteins such as the brush border Na+/glucose cotransporter. The Na+/glucose cotransporter was expressed in Xenopus oocytes, and the changes in cell volume were measured under sugar-transporting and nontransporting conditions. We demonstrate that 260 water molecules are directly coupled to each sugar molecule transported and estimate that in the human intestine this accounts for 5 liters of water absorption per day. Other animal and plant cotransporters such as the Na+/Cl−/γ-aminobutyric acid, Na+/iodide and H+/amino acid transporters are also able to transport water and this suggests that cotransporters play an important role in water homeostasis.

The thiazide-sensitive Na–Cl cotransporter is an aldosterone-induced protein

Although the collecting duct is regarded as the primary site at which mineralocorticoids regulate renal sodium transport in the kidney, recent evidence points to the distal convoluted tubule as a possible site of mineralocorticoid action. To investigate whether mineralocorticoids regulate the expression of the thiazide-sensitive Na–Cl cotransporter (TSC), the chief apical sodium entry pathway of distal convoluted tubule cells, we prepared an affinity-purified, peptide-directed antibody to TSC. On immunoblots, the antibody recognized a prominent 165-kDa band in membrane fractions from the renal cortex but not from the renal medulla. Immunofluorescence immunocytochemistry showed TSC labeling only in distal convoluted tubule cells. Semiquantitative immunoblotting studies demonstrated a large increase in TSC expression in the renal cortex of rats on a low-NaCl diet (207 ± 21% of control diet). Immunofluorescence localization in tissue sections confirmed the strong increase in TSC expression. Treatment of rats for 10 days with a continuous subcutaneous infusion of aldosterone also increased TSC expression (380 ± 58% of controls). Furthermore, 7-day treatment of rats with an orally administered mineralocorticoid, fludrocortisone, increased TSC expression (656 ± 114% of controls). We conclude that the distal convoluted tubule is an important site of action of the mineralocorticoid aldosterone, which strongly up-regulates the expression of TSC.

Characterization of a murine type II sodium-phosphate cotransporter expressed in mammalian small intestine

An isoform of the mammalian renal type II Na/Pi-cotransporter is described. Homology of this isoform to described mammalian and nonmammalian type II cotransporters is between 57 and 75%. Based on major diversities at the C terminus, the new isoform is designed as type IIb Na/Pi-cotransporter. Na/Pi-cotransport mediated by the type IIb cotransporter was studied in oocytes of Xenopus laevis. The results indicate that type IIb Na/Pi-cotransport is electrogenic and in contrast to the renal type II isoform of opposite pH dependence. Expression of type IIb mRNA was detected in various tissues, including small intestine. The type IIb protein was detected as a 108-kDa protein by Western blots using isolated small intestinal brush border membranes and by immunohistochemistry was localized at the luminal membrane of mouse enterocytes. Expression of the type IIb protein in the brush borders of enterocytes and transport characteristics suggest that the described type IIb Na/Pi-cotransporter represents a candidate for small intestinal apical Na/Pi-cotransport.