The Sodium Bicarbonate Cotransporter (NBCe1) Is Essential for Normal Development of Mouse Dentition

Proximal renal tubular acidosis (pRTA) is a syndrome caused by abnormal proximal tubule reabsorption of bicarbonate resulting in metabolic acidosis. Patients with mutations to the SLC4A4 gene (coding for the sodium bicarbonate cotransporter NBCe1), have pRTA, growth delay, ocular defects, and enamel abnormalities. In an earlier report, we provided the first evidence that enamel cells, the ameloblasts, express NBCe1 in a polarized fashion, thereby contributing to trans-cellular bicarbonate transport. To determine whether NBCe1 plays a critical role in enamel development, we studied the expression of NBCe1 at various stages of enamel formation in wild-type mice and characterized the biophysical properties of enamel in NBCe1^−/− animals. The enamel of NBCe1^−/− animals was extremely hypomineralized and weak with an abnormal prismatic architecture. The expression profile of amelogenin, a known enamel-specific gene, was not altered in NBCe1^−/− animals. Our results show for the first time that NBCe1 expression is required for the development of normal enamel. This study provides a mechanistic model to account for enamel abnormalities in certain patients with pRTA.     

Defective coupling of apical PTH receptors to phospholipase C prevents internalization of the Na+-phosphate cotransporter NaPi-IIa in Nherf1-deficient mice

Phosphate reabsorption in the renal proximal tubule occurs mostly via the type IIa Na⁺-phosphate cotransporter (NaP_i-IIa) in the brush border membrane (BBM). The activity and localization of NaP_i-IIa are regulated, among other factors, by parathyroid hormone (PTH). NaP_i-IIa interacts in vitro via its last three COOH-terminal amino acids with the PDZ protein Na⁺/H⁺-exchanger isoform 3 regulatory factor (NHERF)-1 (NHERF1). Renal phosphate reabsorption in Nherf1-deficient mice is altered, and NaP_i-IIa expression in the BBM is reduced. In addition, it has been proposed that NHERF1 and NHERF2 are important for the coupling of PTH receptors (PTHRs) to phospholipase C (PLC) and the activation of the protein kinase C pathway. We tested the role of NHERF1 in the regulation of NaP_i-IIa by PTH in Nherf1-deficient mice. Immunohistochemistry and Western blotting demonstrated that stimulation of apical and basolateral receptors with PTH-(1–34) led to internalization of NaP_i-IIa in wild-type and Nherf1-deficient mice. Stimulation of only apical receptors with PTH-(3–34) failed to induce internalization in Nherf1-deficient mice. Expression and localization of apical PTHRs were similar in wild-type and Nherf1-deficient mice. Activation of the protein kinase C- and A-dependent pathways with 1,2-dioctanoyl-sn-glycerol or 8-bromo-cAMP induced normal internalization of NaP_i-IIa in wild-type, as well as Nherf1-deficient, mice. Stimulation of PLC activity due to apical PTHRs was impaired in Nherf1-deficient mice. These data suggest that NHERF1 in the proximal tubule is important for PTH-induced internalization of NaP_i-IIa and, specifically, couples the apical PTHR to PLC. phosphate cotransporter; PDZ protein; parathyroid hormone; proximal tubule     

Oligomeric structure and minimal functional unit of the electrogenic sodium bicarbonate cotransporter NBCe1-A

The electrogenic sodium bicarbonate cotransporter NBCe1-A mediates the basolateral absorption of sodium and bicarbonate in the proximal tubule. In this study the oligomeric state and minimal functional unit of NBCe1-A were investigated. Wild-type (wt) NBCe1-A isolated from mouse kidney or heterologously expressed in HEK293 cells was predominantly in a dimeric state as was shown using fluorescence energy transfer, pulldown, immunoprecipitation, cross-linking experiments, and nondenaturing perfluorooctanoate-PAGE. NBCe1-A monomers were found to be covalently linked by S-S bonds. When each of the 15 native cysteine residues were individually removed on a wt-NBCe1-A backbone, dimerization of the cotransporter was not affected. In experiments involving multiple native cysteine residue removal, both Cys(630) and Cys(642) in extracellular loop 3 were shown to mediate S-S bond formation between NBCe1-A monomers. When native NBCe1-A cysteine residues were individually reintroduced into a cysteineless NBCe1-A mutant backbone, the finding that a Cys(992) construct that lacked S-S bonds functioned normally indicated that stable covalent linkage of NBCe1-A monomers was not a necessary requirement for functional activity of the cotransporter. Studies using concatameric constructs of wt-NBCe1-A, whose activity is resistant to methanesulfonate reagents, and an NBCe1-A(T442C) mutant, whose activity is completely inhibited by methanesulfonate reagents, confirmed that NBCe1-A monomers are functional. Our results demonstrate that wt-NBCe1-A is predominantly a homodimer, dependent on S-S bond formation that is composed of functionally active monomers.     

Mutagenesis of the N-glycosylation site of hNaSi-1 reduces transport activity

The human Na⁺-sulfate cotransporter (hNaSi-1) belongs to the SLC13 gene family, which also includes the high-affinity Na⁺-sulfate cotransporter (hSUT-1) and the Na⁺-dicarboxylate cotransporters (NaDC). In this study, the location and functional role of the N-glycosylation site of hNaSi-1 were studied using antifusion protein antibodies. Polyclonal antibodies against a glutathione S-transferase fusion protein containing a 65-amino acid peptide of hNaSi-1 (GST-Si65) were raised in rabbits, purified, and then used in Western blotting and immunofluorescence experiments. The antibodies recognized native NaSi-1 proteins in pig and rat brush-border membrane vesicles as well as the recombinant proteins expressed in Xenopus oocytes. Wild-type hNaSi-1 and two N-glycosylation site mutant proteins, N591Y and N591A, were functionally expressed and studied in Xenopus oocytes. The apparent mass of N591Y was not affected by treatment with peptide-N-glycosylase F, in contrast to the mass of wild-type hNaSi-1, which was reduced by up to 15 kDa, indicating that Asn⁵⁹¹ is the N-glycosylation site. Although the cell surface abundance of the two glycosylation site mutants, N591Y and N591A, was greater than that of wild-type hNaSi-1, both mutants had greatly reduced V_max, with no change in K_m. These results suggest that Asn⁵⁹¹ and/or N-glycosylation is critical for transport activity in NaSi-1. antifusion protein antibodies; Xenopus oocytes; sulfate; immunofluorescence     

A novel missense mutation in the sodium bicarbonate cotransporter (NBCe1/SLC4A4) causes proximal tubular acidosis and glaucoma through ion transport defects

In humans and terrestrial vertebrates, the kidney controls systemic pH in part by absorbing filtered bicarbonate in the proximal tubule via an electrogenic Na+/HCO3- cotransporter (NBCe1/SLC4A4). Recently, human genetics revealed that NBCe1 is the major renal contributor to this process. Homozygous point mutations in NBCe1 cause proximal renal tubular acidosis (pRTA), glaucoma, and cataracts (Igarashi, T., Inatomi, J., Sekine, T., Cha, S. H., Kanai, Y., Kunimi, M., Tsukamoto, K., Satoh, H., Shimadzu, M., Tozawa, F., Mori, T., Shiobara, M., Seki, G., and Endou, H. (1999) Nat. Genet. 23, 264-266). We have identified and functionally characterized a novel, homozygous, missense mutation (S427L) in NBCe1, also resulting in pRTA and similar eye defects without mental retardation. To understand the pathophysiology of the syndrome, we expressed wild-type (WT) NBCe1 and S427L-NBCe1 in Xenopus oocytes. Function was evaluated by measuring intracellular pH (HCO3- transport) and membrane currents using microelectrodes. HCO3- -elicited currents for S427L were approximately 10% of WT NBCe1, and CO2-induced acidification was approximately 4-fold faster. Na+ -dependent HCO3- transport (currents and acidification) was also approximately 10% of WT. Current-voltage (I-V) analysis reveals that S427L has no reversal potential in HCO3-, indicating that under physiological ion gradient conditions, NaHCO3 could not move out of cells as is needed for renal HCO3- absorption and ocular pressure homeostasis. I-V analysis without Na+ further shows that the S427L-mediated NaHCO3 efflux mode is depressed or absent. These experiments reveal that voltage- and Na+ -dependent transport by S427L-hkNBCe1 is unfavorably altered, thereby causing both insufficient HCO3- absorption by the kidney (proximal RTA) and inappropriate anterior chamber fluid transport (glaucoma).     

Deciphering PiT transport kinetics and substrate specificity using electrophysiology and flux measurements

Members of the SLC20 family or type III Na⁺-coupled P_i cotransporters (PiT-1, PiT-2) are ubiquitously expressed in mammalian tissue and are thought to perform a housekeeping function for intracellular P_i homeostasis. Previous studies have shown that PiT-1 and PiT-2 mediate electrogenic P_i cotransport when expressed in Xenopus oocytes, but only limited kinetic characterizations were made. To address this shortcoming, we performed a detailed analysis of SLC20 transport function. Three SLC20 clones (Xenopus PiT-1, human PiT-1, and human PiT-2) were expressed in Xenopus oocytes. Each clone gave robust Na⁺-dependent ³²P_i uptake, but only Xenopus PiT-1 showed sufficient activity for complete kinetic characterization by using two-electrode voltage clamp and radionuclide uptake. Transport activity was also documented with Li⁺ substituted for Na⁺. The dependence of the P_i-induced current on P_i concentration was Michaelian, and the dependence on Na⁺ concentration indicated weak cooperativity. The dependence on external pH was unique: the apparent P_i affinity constant showed a minimum in the pH range 6.2–6.8 of 0.05 mM and increased to 0.2 mM at pH 5.0 and pH 8.0. Xenopus PiT-1 stoichiometry was determined by dual ²²Na-³²P_i uptake and suggested a 2:1 Na⁺:P_i stoichiometry. A correlation of ³²P_i uptake and net charge movement indicated one charge translocation per P_i. Changes in oocyte surface pH were consistent with transport of monovalent P_i. On the basis of the kinetics of substrate interdependence, we propose an ordered binding scheme of Na⁺:H₂PO₄^–:Na⁺. Significantly, in contrast to type II Na⁺-P_i cotransporters, the transport inhibitor phosphonoformic acid did not inhibit PiT-1 or PiT-2 activity. Na⁺-P_i cotransport; two-electrode voltage clamp; surface pH electrode; SLC20; retroviral receptor     

NKCC1 and NHE1 are abundantly expressed in the basolateral plasma membrane of secretory coil cells in rat, mouse, and human sweat glands

In isolated sweat glands, bumetanide inhibits sweat secretion. The mRNA encoding bumetanide-sensitive Na⁺-K⁺-Cl^– cotransporter (NKCC) isoform 1 (NKCC1) has been detected in sweat glands; however, the cellular and subcellular protein localization is unknown. Na⁺/H⁺ exchanger (NHE) isoform 1 (NHE1) protein has been localized to both the duct and secretory coil of human sweat duct; however, the NHE1 abundance in the duct was not compared with that in the secretory coil. The aim of this study was to test whether mRNA encoding NKCC1, NKCC2, and Na⁺-coupled acid-base transporters and the corresponding proteins are expressed in rodent sweat glands and, if expressed, to determine the cellular and subcellular localization in rat, mouse, and human eccrine sweat glands. NKCC1 mRNA was demonstrated in rat palmar tissue, including sweat glands, using RT-PCR, whereas NKCC2 mRNA was absent. Also, NHE1 mRNA was demonstrated in rat palmar tissue, whereas NHE2, NHE3, NHE4, electrogenic Na⁺-HCO₃^– cotransporter 1 NBCe1, NBCe2, electroneutral Na⁺-HCO₃^– cotransporter NBCn1, and Na⁺-dependent Cl^–/HCO₃^– exchanger NCBE mRNA were not detected. The expression of NKCC1 and NHE1 proteins was confirmed in rat palmar skin by immunoblotting, whereas NKCC2, NHE2, and NHE3 proteins were not detected. Immunohistochemistry was performed using sections from rat, mouse, and human palmar tissue. Immunoperoxidase labeling revealed abundant expression of NKCC1 and NHE1 in the basolateral domain of secretory coils of rat, mouse, and human sweat glands and low expression was found in the coiled part of the ducts. In contrast, NKCC1 and NHE1 labeling was absent from rat, mouse, and human epidermis. Immunoelectron microscopy demonstrated abundant NKCC1 and NHE1 labeling of the basolateral plasma membrane of mouse sweat glands, with no labeling of the apical plasma membranes or intracellular structures. The basolateral NKCC1 of the secretory coils of sweat glands would most likely account for the observed bumetanide-sensitive NaCl secretion in the secretory coils, and the basolateral NHE1 is likely to be involved in Na⁺-coupled acid-base transport. bumetanide; eccrine glands; immunohistochemistry; immunoblotting     

The abts and sulp families of anion transporters from Caenorhabditis elegans

The slc4 and slc26 gene families encode two distinct groups of gene products that transport HCO₃^– and other anions in mammalian cells. The SLC4 and SLC26 proteins are important contributors to transepithelial movement of fluids and electrolytes and to cellular pH and volume regulation. Herein we describe the cDNA cloning from the nematode Caenorhabditis elegans of four anion bicarbonate transporter (abts) homologs of slc4 cDNA and eight sulfate permease (sulp) homologs of slc26 cDNA. Analysis of transgenic nematode strains carrying promoter::GFP fusions suggests relatively restricted expression patterns for many of these genes. At least three genes are expressed primarily in the intestine, three are expressed primarily in the excretory cell, and one is expressed in both of these polarized cell types. One of the genes is also expressed exclusively in the myoepithelium-like cells of the pharynx. Many of the sulp gene products localize to the basolateral membrane rather than to the apical membrane. Several ABTS and SULP proteins exhibited anion transport function in Xenopus oocytes. The strongest Cl^– transporter among these also mediated Cl^–/HCO₃^– exchange. These findings encourage exploitation of the genetic strengths of the nematode model system in the study of the physiological roles of anion transport by the proteins of these two highly conserved gene families.     

Phosphate transport by the human renal cotransporter NaPi-3 expressed in HEK-293 cells

The human renal Na-PO4cotransporter gene NaPi-3 was expressed in human embryonic kidneyHEK-293 cells, and the transport characteristics were measured in cellstransfected with a vector containing NaPi-3 or with the vector alone(sham transfected). The initial rate of32PO4influx had saturation kinetics for external Na andPO4 with K Na1/2 of 128 mM(PO4 = 0.1 mM) andK PO41/2of 0.084 mM (extracellular Na = 143 mM) in sham- and NaPi-3-transfectedcells expressing the transporter. Transfection had no effect on theNa-independent 32PO4influx, but transfection increased Na-dependent32PO4influxes 2.5- to 5-fold. Of the alkali cations, only Na significantly supported PO4 influx. Arsenateinhibited flux with an inhibition constant of 0.4 mM. The phosphatetransport in sham- and NaPi-3-transfected cells has nearly the sametemperature dependence in the absence and presence of extracellularNa. The Na-dependent phosphate flux decreased with pH insham-transfected cells but was pH independent in transfected cells. TheNa-dependent32PO4influx was inhibited byp-chloromercuriphenylsulfonate,phosphonoformate, phloretin, vanadate, and5-(N-methyl-N-isobutyl)-amiloridebut not by amiloride or other amiloride analogs. These functional characteristics are in general agreement with the known behavior ofNaPi-3 homologues in the renal tubule of other species and, thus,demonstrate the fidelity of this transfection system for the study ofthis protein. Commensurate with the increased functional expression,there was an increase in the amount of NaPi-3 protein by Westernanalysis.

     

Na+-dependent HCO3- uptake into the rat choroid plexus epithelium is partially DIDS sensitive

Several studies suggest the involvement of Na⁺ and HCO₃^– transport in the formation of cerebrospinal fluid. Two Na⁺-dependent HCO₃^– transporters were recently localized to the epithelial cells of the rat choroid plexus (NBCn1 and NCBE), and the mRNA for a third protein was also detected (NBCe2) (Praetorius J, Nejsum LN, and Nielsen S. Am J Physiol Cell Physiol 286: C601–C610, 2004). Our goal was to immunolocalize the NBCe2 to the choroid plexus by immunohistochemistry and immunogold electronmicroscopy and to functionally characterize the bicarbonate transport in the isolated rat choroid plexus by measurements of intracellular pH (pH_i) using a dual-excitation wavelength pH-sensitive dye (BCECF). Both antisera derived from COOH-terminal and NH₂-terminal NBCe2 peptides localized NBCe2 to the brush-border membrane domain of choroid plexus epithelial cells. Steady-state pH_i in choroidal cells increased from 7.03 ± 0.02 to 7.38 ± 0.02 (n = 41) after addition of CO₂/HCO₃^– into the bath solution. This increase was Na⁺ dependent and inhibited by the Cl^– and HCO₃^– transport inhibitor DIDS (200 µM). This suggests the presence of Na⁺-dependent, partially DIDS-sensitive HCO₃^– uptake. The pH_i recovery after acid loading revealed an initial Na⁺ and HCO₃^–-dependent net base flux of 0.828 ± 0.116 mM/s (n = 8). The initial flux in the presence of CO₂/HCO₃^– was unaffected by DIDS. Our data support the existence of both DIDS-sensitive and -insensitive Na⁺- and HCO₃^–-dependent base loader uptake into the rat choroid plexus epithelial cells. This is consistent with the localization of the three base transporters NBCn1, Na⁺-driven Cl^– bicarbonate exchanger, and NBCe2 in this tissue. bicarbonate metabolism; BCECF; cerebrospinal fluid; acid/base transport; ammonium prepulse     

Endogenous expression of the renal high-affinity H+-peptide cotransporter in LLC-PK1 cells

The reabsorption of filtered di- andtripeptides as well as certain peptide mimetics from the tubular lumeninto renal epithelial cells is mediated by anH⁺-coupledhigh-affinity transport process. Here we demonstrate for the first timeH⁺-coupled uptake of dipeptidesinto the renal proximal tubule cell lineLLC-PK₁. Transport was assessed1) by uptake studies using theradiolabeled dipeptideD-[³H]Phe-L-Ala,2) by cellular accumulation of the fluorescent dipeptide D-Ala-Lys-AMCA, and3) by measurement of intracellularpH (pH_i) changes as aconsequence of H⁺-coupleddipeptide transport. Uptake ofD-Phe-L-Alaincreased linearly over 11 days postconfluency and showed all thecharacteristics of the kidney cortex high-affinity peptide transporter,e.g., a pH optimum for transport ofD-Phe-L-Alaof 6.0, an apparent K_m value forinflux of 25.8 ± 3.6 µM, and affinities of differently chargeddipeptides or the -lactam antibiotic cefadroxil to the binding sitein the range of 20-80 µM.pH_i measurements established thepeptide transporter to induce pronounced intracellular acidification inLLC-PK₁ cells and confirm itspostulated role as a cellular acid loader.

     

Role of NBC1 in apical and basolateral HCO3- permeabilities and transendothelial HCO3- fluxes in bovine corneal endothelium

Corneal transparency and hydration control are dependent on HCO₃^– transport properties of the corneal endothelium. Recent work (13) suggested the presence of an apical 1Na⁺-3HCO₃^– cotransporter (NBC1) in addition to a basolateral 1Na⁺-2HCO₃^– cotransporter. We examined whether the NBC1 cotransporter contributes significantly to basolateral or apical HCO₃^– permeability and whether the cotransporter participates in transendothelial net HCO₃^– flux in cultured bovine corneal endothelium. NBC1 protein expression was reduced using small interfering RNA (siRNA). Immunoblot analysis showed that 5–15 nM siRNA decreased NBC1 expression by 80–95%, 4 days posttransfection. Apical and basolateral HCO₃^– permeabilities were determined by measuring the rate of pH_i change when HCO₃^– was removed from the bath under constant pH or constant CO₂ conditions. Using either protocol, we found that cultures treated with NBC1 siRNA had sixfold lower basolateral HCO₃^– permeability than untreated or siCONTROL siRNA-treated cells. Apical HCO₃^– permeability was unaffected by NBC1 siRNA treatment. Net non-steady-state HCO₃^– flux was 0.707 ± 0.009 mM·min^–1·cm² in the basolateral-to-apical direction and increased to 1.74 ± 0.15 when cells were stimulated with 2 µM forskolin. Treatment with 5 nM siRNA decreased basolateral-to-apical flux by 67%, whereas apical-to-basolateral flux was unaffected, significantly decreasing net HCO₃^– flux to 0.236 ± 0.002. NBC1 siRNA treatment or 100 µM ouabain also eliminated steady-state HCO₃^– flux, as measured by apical compartment alkalinization. Collectively, reduced basolateral HCO₃^– permeability, basolateral-to-apical fluxes, and net HCO₃^– flux as a result of reduced expression of NBC1 indicate that NBC1 plays a key role in transendothelial HCO₃^– flux and is functional only at the basolateral membrane. corneal endothelium; sodium bicarbonate cotransporter; small interfering RNA; bicarbonate transport     

Glutamate 59 is critical for transport function of the amino acid cotransporter KAAT1

KAAT1 is a neutral amino acid transporter activated by K⁺ or by Na⁺ (9). The protein shows significant homology with members of the Na⁺/Cl^–-dependent neurotransmitter transporter super family. E59G KAAT1, expressed in Xenopus oocytes, exhibited a reduced leucine uptake [20–30% of wild-type (WT)], and kinetic analysis indicated that the loss of activity was due to reduction of V_max and apparent affinity for substrates. Electrophysiological analysis revealed that E59G KAAT1 has presteady-state and uncoupled currents larger than WT but no leucine-induced currents. Site-directed mutagenesis analysis showed the requirement of a negative charge in position 59 of KAAT1. The analysis of permeant and impermeant methanethiosulfonate reagent effects confirmed the intracellular localization of glutamate 59. Because the 2-aminoethyl methanethiosulfonate hydrobromid inhibition was not prevented by the presence of Na⁺ or leucine, we concluded that E59 is not directly involved in the binding of substrates. N-ethylmaleimide inhibition was qualitatively and quantitatively different in the two transporters, WT and E59G KAAT1, having the same cysteine residues. This indicates an altered accessibility of native cysteine residues due to a modified spatial organization of E59G KAAT1. The arginine modifier phenylglyoxal effect supports this hypothesis: not only cysteine but also arginine residues become more accessible to the modifying reagents in the mutant E59G. In conclusion, the results presented indicate that glutamate 59 plays a critical role in the three-dimensional organization of KAAT1. amino acid transport; structure/function; amino acid modifiers; Manduca sexta     

Membrane cholesterol extraction decreases Na+ transport in A6 renal epithelia

In this study, we have investigated the dependence of Na⁺ transport regulation on membrane cholesterol content in A6 renal epithelia. We continuously monitored short-circuit current (I_sc), transepithelial conductance (G_T), and transepithelial capacitance (C_T) to evaluate the effects of cholesterol extraction from the apical and basolateral membranes in steady-state conditions and during activation with hyposmotic shock, oxytocin, and adenosine. Cholesterol extraction was achieved by perfusing the epithelia with methyl--cyclodextrin (mCD) for 1 h. In steady-state conditions, apical membrane cholesterol extraction did not significantly affect the electrophysiological parameters; in contrast, marked reductions were observed during basolateral mCD treatment. However, apical mCD application hampered the responses of I_sc and G_T to hypotonicity, oxytocin, and adenosine. Analysis of the blocker-induced fluctuation in I_sc demonstrated that apical mCD treatment decreased the epithelial Na⁺ channel (ENaC) open probability (P_o) in the steady state as well as after activation of Na⁺ transport by adenosine, whereas the density of conducting channels was not significantly changed as confirmed by C_T measurements. Na⁺ transport activation by hypotonicity was abolished during basolateral mCD treatment as a result of reduced Na⁺/K⁺ pump activity. On the basis of the findings in this study, we conclude that basolateral membrane cholesterol extraction reduces Na⁺/K⁺ pump activity, whereas the reduced cholesterol content of the apical membranes affects the activation of Na⁺ transport by reducing ENaC P_o. epithelial Na⁺ channel; Na⁺-K⁺-ATPase activity; short-circuit current; methyl--cyclodextrin; channel open probability     

Volume sensitivity of cation-Cl- cotransporters is modulated by the interaction of two kinases: Ste20-related proline-alanine-rich kinase and WNK4

In the present study, we have demonstrated functional interaction between Ste20-related proline-alanine-rich kinase (SPAK), WNK4 [with no lysine (K)], and the widely expressed Na⁺-K⁺-2Cl^– cotransporter type 1 (NKCC1). NKCC1 function, which we measured in Xenopus laevis oocytes under both isosmotic (basal) and hyperosmotic (stimulated) conditions, was unaffected when SPAK and WNK4 were expressed alone. In contrast, expression of both kinases with NKCC1 resulted in a significant increase in cotransporter activity and an insensitivity to external osmolarity or cell volume. NKCC1 activation is dependent on the catalytic activity of SPAK and likely also of WNK4, because mutations in their catalytic domains result in an absence of cotransporter stimulation. The results of our yeast two-hybrid experiments suggest that WNK4 does not interact directly with NKCC1 but does interact with SPAK. Functional experiments demonstrated that the binding of SPAK to WNK4 was also required because a SPAK-interaction-deficient WNK4 mutant (Phe997Ala) did not increase NKCC1 activity. We also have shown that the transport function of K⁺-Cl^– cotransporter type 2 (KCC2), a neuron-specific KCl cotransporter, was diminished by the expression of both kinases under both isosmotic and hyposmotic conditions. Our data are consistent with WNK4 interacting with SPAK, which in turn phosphorylates and activates NKCC1 and phosphorylates and deactivates KCC2. bumetanide; Na⁺-K⁺-2Cl^– cotransporter; K⁺-Cl^– cotransporter; Xenopus oocytes     

Oxalate inhibits renal proximal tubule cell proliferation via oxidative stress, p38 MAPK/JNK, and cPLA2 signaling pathways

Exposure of renal proximal tubule cells to oxalate may play an important role in cell proliferation, but the signaling pathways involved in this effect have not been elucidated. Thus the present study was performed to examine the effect of oxalate on ³H-labeled thymidine incorporation and its related signal pathway in primary cultured rabbit renal proximal tubule cells (PTCs). The effects of oxalate on [³H]thymidine incorporation, lactate dehydrogenase (LDH) release, Trypan blue exclusion, H₂O₂ release, activation of mitogen-activated protein kinases (MAPKs), and ³H-labeled arachidonic acid (AA) release were examined in primary cultured renal PTCs. Oxalate inhibited [³H]thymidine incorporation in a time- and dose-dependent manner. However, its analogs did not affect [³H]thymidine incorporation. Oxalate (1 mM) significantly increased H₂O₂ release, which was blocked by N-acetyl-L-cysteine (NAC) and catalase (antioxidants). Oxalate significantly increased p38 MAPK and stress-activated protein kinase (SAPK)/c-Jun NH₂-terminal kinase (JNK) activity, not p44/42 MAPK. Oxalate stimulated [³H]AA release and translocation of cytosolic phospholipase A₂ (cPLA₂) from the cytosolic fraction to the membrane fraction. Indeed, oxalate significantly increased prostaglandin E₂ (PGE₂) production compared with control. Oxalate-induced inhibition of [³H]thymidine incorporation and increase of [³H]AA release were prevented by antioxidants (NAC), a p38 MAPK inhibitor (SB-203580), a SAPK/JNK inhibitor (SP-600125), or PLA₂ inhibitors [mepacrine and arachidonyl trifluoromethyl ketone (AACOCF₃)], but not by a p44/42 MAPK inhibitor (PD-98059). These findings suggest that oxalate inhibits renal PTC proliferation via oxidative stress, p38 MAPK/JNK, and cPLA₂ signaling pathways. kidney; mitogen-activated protein kinase; phospholipase A₂     

Riboflavin transport by isolated perfused rabbit renal proximal tubules

Rabbit renal proximal tubular transport of riboflavin(RF) was examined by using the in vitro isolated tubule perfusiontechnique. We found that proximal tubules actively reabsorbed(J_lb) and secreted (J_bl)RF. At 0.1 µM RF concentration, J_bl wassignificantly higher than J_lb, resulting in anet secretion. This net secretion of RF was decreased at 0.01 µM RFconcentration and increased at 1 µM RF concentration. BothJ_lb and J_bl wereinhibited by lowering temperature or by adding iodoacetate, a metabolicinhibitor, and lumichrome, an RF analog, suggesting the involvement ofcarrier-mediated transport mechanisms. J_bl wasinhibited by probenecid, an anion transport inhibitor, and bypara-aminohippuric acid, an organic anion, suggesting therelevance of RF secretion to renal organic anion transport.J_bl was also inhibited by alkaline pH (8.0) and by the calmodulin inhibitor trifluoperazine, indicating the influence of pH and Ca²⁺/calmodulin-dependent pathway on RFsecretion. Finally, we found that addition of chlorpromazine, aphenothiazine derivative, inhibited both J_lb andJ_bl, raising the concern about the nutritionalstatus in patients receiving such a type of medication.

     

Effect of PCMBS on CO2 permeability of Xenopus oocytes expressing aquaporin 1or its C189S mutant

A recent study onXenopus oocytes [N. L. Nakhoul,M. F. Romero, B. A. Davis, and W. F. Boron. Am. J. Physiol. 274 (CellPhysiol. 43): C543-548, 1998] injected withcarbonic anhydrase showed that expressing aquaporin 1 (AQP1) increasesby ~40% the rate at which exposing the cell toCO₂ causes intracellular pH tofall. This observation is consistent with several interpretations.Overexpressing AQP1 might increase apparentCO₂ permeability by1) allowingCO₂ to pass through AQP1,2) stimulating injected carbonicanhydrase, 3) enhancing theCO₂ solubility of the membrane'slipid, or 4) increasing theexpression of a native "gas channel." The purpose of the presentstudy was to distinguish among these possibilities. We found thatexpressing the H₂O channel AQP1 inXenopus oocytes increases theCO₂ permeability of oocytes in anexpression-dependent fashion, whereas expressing theK⁺ channel ROMK1 has no effect.The mercury derivativep-chloromercuriphenylsulfonic acid(PCMBS), which inhibits the H₂Omovement through AQP1, also blocks the AQP1-dependent increase inCO₂ permeability. Themercury-insensitive C189S mutant of AQP1 increases theCO₂ permeability of the oocyte tothe same extent as does the wild-type channel. However, the C189S-dependent increase in CO₂permeability is unaffected by treatment with PCMBS. These data rule outoptions 2-4 listed above. Thusour results suggest that CO₂passes through the pore of AQP1 and are the first data to demonstratethat a gas can enter a cell by a means other than diffusing through themembrane lipid.

     

Localization of Na+-HCO-3 cotransporter (NBC-1) variants in rat and human pancreas

Mutations inNa⁺-HCO cotransporter (NBC-1) causeproximal renal tubular acidosis (pRTA) associated with ocularabnormalities. One pRTA patient had increased serum amylase, suggestingpossible evidence of pancreatitis. To further delineate a link betweenNBC-1 inactivation and pancreatic dysfunction, immunohistochemicalanalysis was performed on rat and human pancreas using antibodiesagainst kidney-type (kNBC-1) and pancreatic-type (pNBC-1) transporters.In rat pancreas, the anti-pNBC-1 antibody labeled acinar cells and bothapical and basolateral membranes of medium and large duct cells. Inhuman pancreas, on the other hand, the anti-pNBC-1 antibody did notlabel acinar cells, although it did label the basolateral membranes ofthe entire duct system. The labeling by anti-kNBC-1 antibody wasdetected in only a limited number of rat pancreatic duct cells. Toexamine the effects of pRTA-related mutations, R342S and R554H, onpNBC-1 function, we performed functional analysis and found that bothmutants had reduced transport activities compared with the wild-typepNBC-1. These results indicate that pNBC-1 is the predominant variant that mediates basolateral HCO uptake into duct cellsin both rat and human pancreas. The loss of pNBC-1 function ispredicted to have significant impact on overall ductal HCO secretion, which could potentially lead topancreatic dysfunction.