NBCe2 (Slc4a5) Is Expressed in the Renal Connecting Tubules and Cortical Collecting Ducts and Mediates Base Extrusion.

Arterial hypertension, is a common disorder with multiple and variable etiologies. Single nucleotide polymorphism analyses have detected an association between variants in the gene encoding the electrogenic Na+:HCO3 - cotransporter NBCe2 (Slc4a5), and salt-sensitive hypertension. Mice with genetic deletion of NBCe2 are hypertensive, and the cause of the blood pressure (BP) increase is believed to arise from a lack of renal NBCe2 function. The exact cellular expression of NBCe2 in the kidney tubular system is, however, not determined. Here, we find NBCe2 to be expressed predominantly in isolated connecting tubules (CNT) and cortical collecting ducts (CD) by RT-PCR. In isolated renal CNT and CCD, genetic deletion of NBCe2 leads to decreased net base extrusion. To determine the role of renal NBCe2 in the development of hypertension, we generated CNT and intercalated cell NBCe2 knockout mice by crossing an Slc4a5 lox mouse with mice expressing cre recombinase under the V-ATPase B1 subunit promotor. Although the mice displayed changes in the expression of renal membrane transporters, we did not detect hypertension in these mice by tail cuff recordings. In conclusion, while global NBCe2 deletion certainly causes hypertension this study cannot confirm the role of renal NBCe2 expression in blood pressure regulation.

The choroid plexus sodium-bicarbonate cotransporter NBCe2 regulates mouse cerebrospinal fluid pH.

KEY POINTS: Normal pH is crucial for proper functioning of the brain, and disorders increasing the level of CO2 in the blood lead to a decrease in brain pH. CO2 can easily cross the barriers of the brain and will activate chemoreceptors leading to an increased exhalation of CO2 . The low pH, however, is harmful and bases such as HCO3- are imported across the brain barriers in order to normalize brain pH. We show that the HCO3- transporter NBCe2 in the choroid plexus of the blood-cerebrospinal fluid barrier is absolutely necessary for normalizing CSF pH during high levels of CO2 . This discovery represents a significant step in understanding the molecular mechanisms behind regulation of CSF pH during acid-base disturbances, such as chronic lung disease. ABSTRACT: The choroid plexus epithelium (CPE) is located in the brain ventricles where it produces the majority of the cerebrospinal fluid (CSF). The hypothesis that normal brain function is sustained by CPE-mediated CSF pH regulation by extrusion of acid-base equivalents was tested by determining the contribution of the electrogenic Na+ -HCO3- cotransporter NBCe2 to CSF pH regulation. A novel strain of NBCe2 (Slc4a5) knockout (KO) mice was generated and validated. The base extrusion rate after intracellular alkalization was reduced by 77% in NBCe2 KO mouse CPE cells compared to control mice. NBCe2 KO mice and mice with CPE-targeted NBCe2 siRNA knockdown displayed a reduction in CSF pH recovery during hypercapnia-induced acidosis of approximately 85% and 90%, respectively, compared to control mice. NBCe2 KO did not affect baseline respiration rate or tidal volume, and the NBCe2 KO and wild-type (WT) mice displayed similar ventilatory responses to 5% CO2 exposure. NBCe2 KO mice were not protected against pharmacological or heating-induced seizure development. In conclusion, we establish the concept that the CPE is involved in the regulation of CSF pH by demonstrating that NBCe2 is necessary for proper CSF pH recovery after hypercapnia-induced acidosis.

The murine choroid plexus epithelium expresses the 2Cl-/H+ exchanger ClC-7 and Na+/H+ exchanger NHE6 in the luminal membrane domain.

The choroid plexus epithelium within the brain ventricles secretes the majority of the cerebrospinal fluid (CSF). The luminal Na+-K+-ATPase acts in concert with a host of other transport proteins to mediate efficient fluid secretion across the epithelium. The CSF contains little protein buffer, but the pH value seems nonetheless maintained within narrow limits, even when faced with acid-base challenges. The involvement of choroid plexus acid-base transporters in CSF pH regulation is highlighted by the expression of several acid-base transporters in the epithelium. The aim of the present study was to identify novel acid-base transporters expressed in the luminal membrane of the choroid plexus epithelium to pave the way for systematic investigations of each candidate transporter in the regulation of CSF pH. Mass spectrometry analysis of proteins from epithelial cells isolated by fluorescence-activated cell sorting identified the Cl-/H+ exchangers ClC-3, -4, -5, and -7 in addition to known choroid plexus acid-base transporters. RT-PCR on FACS isolated epithelial cells confirmed the expression of the corresponding mRNAs, as well as Na+/H+ exchanger NHE6 mRNA. Both NHE6 and ClC-7 were immunolocalized to the luminal plasma membrane domain of the choroid plexus epithelial cells. Dynamic imaging of intracellular pH and membrane potential changes in isolated choroid plexus epithelial cells demonstrated Cl- gradient-driven changes in intracellular pH and membrane potential that are consistent with Cl-/H+ exchange. In conclusion, we have detected for the first time NHE6 and ClC-7 in the choroid plexus, which are potentially involved in pH regulation of the CSF.

The V-ATPase is expressed in the choroid plexus and mediates cAMP-induced intracellular pH alterations.

The cerebrospinal fluid (CSF) pH influences brain interstitial pH and, therefore, brain function. We hypothesized that the choroid plexus epithelium (CPE) expresses the vacuolar H+-ATPase (V-ATPase) as an acid extrusion mechanism in the luminal membrane to counteract detrimental elevations in CSF pH. The expression of mRNA corresponding to several V-ATPase subunits was demonstrated by RT-PCR analysis of CPE cells (CPECs) isolated by fluorescence-activated cell sorting. Immunofluorescence and electron microscopy localized the V-ATPase primarily in intracellular vesicles with only a minor fraction in the luminal microvillus area. The vesicles did not translocate to the luminal membrane in two in vivo models of hypocapnia-induced alkalosis. The Na+-independent intracellular pH (pHi) recovery from acidification was studied in freshly isolated clusters of CPECs. At extracellular pH (pHo) 7.4, the cells failed to display significant concanamycin A-sensitive pHi recovery (i.e., V-ATPase activity). The recovery rate in the absence of Na+ amounted to <10% of the pHi recovery rate observed in the presence of Na+ Recovery of pHi was faster at pHo 7.8 and was abolished at pHo 7.0. The concanamycin A-sensitive pHi recovery was stimulated by cAMP at pH 7.4 in vitro, but intraventricular infusion of the membrane-permeant cAMP analog 8-CPT-cAMP did not result in trafficking of the V-ATPase. In conclusion, we find evidence for the expression of a minor fraction of V-ATPase in the luminal membrane of CPECs. This fraction does not contribute to enhanced acid extrusion at high extracellular pH, but seems to be activated by cAMP in a trafficking-independent manner.

Na(+) dependent acid-base transporters in the choroid plexus; insights from slc4 and slc9 gene deletion studies.

The choroid plexus epithelium (CPE) is located in the ventricular system of the brain, where it secretes the majority of the cerebrospinal fluid (CSF) that fills the ventricular system and surrounds the central nervous system. The CPE is a highly vascularized single layer of cuboidal cells with an unsurpassed transepithelial water and solute transport rate. Several members of the slc4a family of bicarbonate transporters are expressed in the CPE. In the basolateral membrane the electroneutral Na(+) dependent Cl(-)/HCO3 (-) exchanger, NCBE (slc4a10) is expressed. In the luminal membrane, the electrogenic Na(+):HCO3 (-) cotransporter, NBCe2 (slc4a5) is expressed. The electroneutral Na(+):HCO3 (-) cotransporter, NBCn1 (slc4a7), has been located in both membranes. In addition to the bicarbonate transporters, the Na(+)/H(+) exchanger, NHE1 (slc9a1), is located in the luminal membrane of the CPE. Genetically modified mice targeting slc4a2, slc4a5, slc4a7, slc4a10, and slc9a1 have been generated. Deletion of slc4a5, 7 or 10, or slc9a1 has numerous impacts on CP function and structure in these mice. Removal of the transporters affects brain ventricle size (slc4a5 and slc4a10) and intracellular pH regulation (slc4a7 and slc4a10). In some instances, removal of the proteins from the CPE (slc4a5, 7, and 10) causes changes in abundance and localization of non-target transporters known to be involved in pH regulation and CSF secretion. The focus of this review is to combine the insights gathered from these knockout mice to highlight the impact of slc4 gene deletion on the CSF production and intracellular pH regulation resulting from the deletion of slc4a5, 7 and 10, and slc9a1. Furthermore, the review contains a comparison of the described human mutations of these genes to the findings in the knockout studies. Finally, the future perspective of utilizing these proteins as potential targets for the treatment of CSF disorders will be discussed.