Cerebrospinal fluid pH regulation.

The cerebrospinal fluid (CSF) fills the brain ventricles and the subarachnoid space surrounding the brain and spinal cord. The fluid compartment of the brain ventricles communicates with the interstitial fluid of the brain across the ependyma. In comparison to blood, the CSF contains very little protein to buffer acid-base challenges. Nevertheless, the CSF responds efficiently to changes in systemic pH by mechanisms that are dependent on the CO2/HCO3- buffer system. This is evident from early studies showing that the CSF secretion is sensitive to inhibitors of acid/base transporters and carbonic anhydrase. The CSF is primarily generated by the choroid plexus, which is a well-vascularized structure arising from the pial lining of the brain ventricles. The epithelial cells of the choroid plexus host a range of acid/base transporters, many of which participate in CSF secretion and most likely contribute to the transport of acid/base equivalents into the ventricles. This review describes the current understanding of the molecular mechanisms in choroid plexus acid/base regulation and the possible role in CSF pH regulation.

The Cyst Epithelium in Polycystic Kidney Disease Patients Displays Normal Apical-Basolateral Cell Polarity.

The main characteristic of polycystic kidney disease is the development of multiple fluid-filled renal cysts. The discovery of mislocalized sodium-potassium pump (Na,K-ATPase) in the apical membrane of cyst-lining epithelia alluded to reversal of polarity as a possible explanation for the fluid secretion. The topic of apical Na,K-ATPase in cysts remains controversial. We investigated the localization of the Na,K-ATPase and assessed the apical-basolateral polarization of cyst-lining epithelia by means of immunohistochemistry in kidney tissue from six polycystic kidney disease patients undergoing nephrectomy. The Na,K-ATPase α1 subunit was conventionally situated in the basolateral membrane of all immunoreactive cysts. Proteins of the Crumbs and partitioning defective (Par) complexes were localized to the apical membrane domain in cyst epithelial cells. The apical targeting protein Syntaxin-3 also immunolocalized to the apical domain of cyst-lining epithelial cells. Proteins of the basolateral Scribble complex immunolocalized to the basolateral domain of cysts. Thus, no deviations from the typical epithelial distribution of basic cell polarity proteins were observed in the cysts from the six patients. Furthermore, we confirmed that cysts can originate from virtually any tubular segment with preserved polarity. In conclusion, we find no evidence of a reversal in apical-basolateral polarity in cyst-lining epithelia in polycystic kidney disease.

Multiple Na,K-ATPase Subunits Colocalize in the Brush Border of Mouse Choroid Plexus Epithelial Cells.

(1) Background: The unusual accumulation of Na,K-ATPase complexes in the brush border membrane of choroid plexus epithelial cells have intrigued researchers for decades. However, the full range of the expressed Na,K-ATPase subunits and their relation to the microvillus cytoskeleton remains unknown. (2) Methods: RT-PCR analysis, co-immunoprecipitation, native PAGE, mass spectrometry, and differential centrifugation were combined with high-resolution immunofluorescence histochemistry, proximity ligase assays, and stimulated emission depletion (STED) microscopy on mouse choroid plexus cells or tissues in order to resolve these issues. (3) Results: The choroid plexus epithelium expresses Na,K-ATPase subunits α1, α2, ß1, ß2, ß3, and phospholemman. The α1, α2, ß1, and ß2, subunits are all localized to the brush border membrane, where they appear to form a complex. The ATPase complexes may stabilize in the brush border membrane via anchoring to microvillar actin indirectly through ankyrin-3 or directly via other co-precipitated proteins. Aquaporin 1 (AQP1) may form part of the proposed multi-protein complexes in contrast to another membrane protein, the Na-K-2Cl cotransporter 1 (NKCC1). NKCC1 expression seems necessary for full brush border membrane accumulation of the Na,K-ATPase in the choroid plexus. (4) Conclusion: A multitude of Na,K-ATPase subunits form molecular complexes in the choroid plexus brush border, which may bind to the cytoskeleton by various alternative actin binding proteins.

Cerebrospinal fluid secretion by the choroid plexus.

The choroid plexus epithelium is a cuboidal cell monolayer, which produces the majority of the cerebrospinal fluid. The concerted action of a variety of integral membrane proteins mediates the transepithelial movement of solutes and water across the epithelium. Secretion by the choroid plexus is characterized by an extremely high rate and by the unusual cellular polarization of well-known epithelial transport proteins. This review focuses on the specific ion and water transport by the choroid plexus cells, and then attempts to integrate the action of specific transport proteins to formulate a model of cerebrospinal fluid secretion. Significant emphasis is placed on the concept of isotonic fluid transport across epithelia, as there is still surprisingly little consensus on the basic biophysics of this phenomenon. The role of the choroid plexus in the regulation of fluid and electrolyte balance in the central nervous system is discussed, and choroid plexus dysfunctions are described in a very diverse set of clinical conditions such as aging, Alzheimer's disease, brain edema, neoplasms, and hydrocephalus. Although the choroid plexus may only have an indirect influence on the pathogenesis of these conditions, the ability to modify epithelial function may be an important component of future therapies.

Choroid plexus epithelial cells express the adhesion protein P-cadherin at cell-cell contacts and syntaxin-4 in the luminal membrane domain.

The choroid plexus epithelial cells (CPECs) belong to a small group of polarized cells, where the Na+-K+-ATPase is expressed in the luminal membrane. The basic polarity of the cells is, therefore, still debated. We investigated the subcellular distribution of an array of proteins known to play fundamental roles either in establishing and maintaining basic cell polarity or in the polarized delivery and recycling of plasma membrane proteins. Immunofluorescence histochemical analysis was applied to determine the subcellular localization of apical and basolateral membrane determinants. Mass spectrometry analysis of CPECs isolated by fluorescence-activated cell sorting was applied to determine the expression of specific forms of the proteins. CPECs mainly express the cell-adhesive P-cadherin, which is localized to the lateral membranes. Proteins belonging to the Crumbs and partitioning defective (Par) protein complexes were all localized to the luminal membrane domain. Par-1 and the Scribble complex were localized to the basolateral membrane domain. Lethal(2) giant larvae homolog 2 (Lgl2) labeling was preferentially observed in the luminal membrane domain. Phosphatidylinositol 3,4,5-trisphosphate (PIP3) was immunolocalized to the basolateral membrane domain, while phosphatidylinositol 4,5-bisphosphate (PIP2) staining was most prominent in the luminal membrane domain along with the PIP3 phosphatase, Pten. The apical target-SNARE syntaxin-3 and the basolateral target-SNARE syntaxin-4 were both localized to the apical membrane domain in CPECs, which lack cellular expression of the clathrin adaptor protein AP-1B for basolateral protein recycling. In conclusion, the CPECs are conventionally polarized, but express P-cadherin at cell-cell contacts, and Lgl2 and syntaxin-4 in the luminal plasma membrane domain.

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 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.

Adenylyl cyclase 6 is required for maintaining acid-base homeostasis.

Adenylyl cyclase (AC) isoform 6 (AC6) is highly expressed throughout the renal tubule and collecting duct (CD), catalyzes the synthesis of cAMP and contributes to various aspects of renal transport. Several proteins involved in acid-base homeostasis are regulated by cAMP. In the present study, we assess the relative contribution of AC6 to overall acid-base regulation using mice with global deletion of AC6 (AC6-/-) or newly generated mice lacking AC6 in the renal tubule and CD (AC6loxloxPax8Cre). Higher energy expenditure in AC6-/- relative to wild-type (WT) mice, was associated with lower urinary pH, mild alkalosis in conjunction with elevated blood HCO3- concentrations, and significantly higher renal abundance of the H+-ATPase B1 subunit. In contrast with WT mice, AC6-/- mice have a less pronounced increase in urinary pH after 8 days of HCO3- challenge, which is associated with increased blood pH and HCO3- concentrations. Immunohistochemistry demonstrated that AC6 was expressed in intercalated cells (IC), but subcellular distribution of the H+-ATPase B1 subunit, pendrin, and the anion exchangers 1 and 2 in AC6-/- mice was normal. In the AC6-/- mice, H+-ATPase B1 subunit levels after HCO3- challenge were greater, which correlated with a higher number of type A IC. In contrast with the AC6-/- mice, AC6loxloxPax8Cre mice had normal urinary pH under baseline conditions but higher blood HCO3- than controls after HCO3- challenge. In conclusion, AC6 is required for maintaining normal acid-base homeostasis and energy expenditure. Under baseline conditions, renal AC6 is redundant for acid-base balance but becomes important under alkaline conditions.

Isolating subpopulations of human epidermal basal cells based on polyclonal serum against trypsin-resistant CSPG4 epitopes.

Chondroitin sulfate proteoglycan 4 (CSPG4) is highly expressed by human epidermal keratinocytes located at the tip of the dermal papilla where keratinocytes show characteristics of stem cells. However, since available antibodies to CSPG4 are directed against trypsin-sensitive epitopes we have been unable to study these keratinocytes isolated directly from skin samples by flow cytometry. By choosing epitopes of CSPG4 relatively close to the cell membrane we were able to generate a polyclonal antibody that successfully detects CSPG4 on keratinocytes after trypsinization. Although CSPG4-positive basal cells express higher levels of Itgß1 the colony-forming efficiency is slightly lower than CSPG4-negative basal cells. Sorting the directly isolated keratinocytes based on Itgß1 did not reveal differences in colony-forming efficiency between keratinocytes expressing high or low levels of Itgß1. However, after the first passage Itgß1 could be used to predict colony-forming efficiency whether the culture was established from CSPG4-positive or CSPG4-negative basal cell keratinocytes. Although we were unable to detect differences in the colony-forming assay, global gene expression profiling showed that CSPG4-positive basal cell keratinocytes are distinct from CSPG4-negative basal cell keratinocytes. Our study demonstrates that it is possible to generate antibodies against trypsin-resistant epitopes of CSPG4. Our study also documents a marked change in behaviour upon cell culturing and challenges the way we assess for stemness within the human epidermal basal layer.

Transport across the choroid plexus epithelium.

The choroid plexus epithelium is a secretory epithelium par excellence. However, this is perhaps not the most prominent reason for the massive interest in this modest-sized tissue residing inside the brain ventricles. Most likely, the dominant reason for extensive studies of the choroid plexus is the identification of this epithelium as the source of the majority of intraventricular cerebrospinal fluid. This finding has direct relevance for studies of diseases and conditions with deranged central fluid volume or ionic balance. While the concept is supported by the vast majority of the literature, the implication of the choroid plexus in secretion of the cerebrospinal fluid was recently challenged once again. Three newer and promising areas of current choroid plexus-related investigations are as follows: 1) the choroid plexus epithelium as the source of mediators necessary for central nervous system development, 2) the choroid plexus as a route for microorganisms and immune cells into the central nervous system, and 3) the choroid plexus as a potential route for drug delivery into the central nervous system, bypassing the blood-brain barrier. Thus, the purpose of this review is to highlight current active areas of research in the choroid plexus physiology and a few matters of continuous controversy.

Reducing αENaC expression in the kidney connecting tubule induces pseudohypoaldosteronism type 1 symptoms during K+ loading.

Genetic inactivation of the epithelial Na(+) channel α-subunit (αENaC) in the renal collecting duct (CD) does not interfere with Na(+) and K(+) homeostasis in mice. However, inactivation in the CD and a part of the connecting tubule (CNT) induces autosomal recessive pseudohypoaldosteronism type 1 (PHA-1) symptoms in subjects already on a standard diet. In the present study, we further examined the importance of αENaC in the CNT. Knockout mice with αENaC deleted primarily in a part of the CNT (CNT-KO) were generated using Scnn1a(lox/lox) mice and Atp6v1b1::Cre mice. With a standard diet, plasma Na(+) concentration ([Na(+)]) and [K(+)], and urine Na(+) and K(+) output were unaffected. Seven days of Na(+) restriction (0.01% Na(+)) led to a higher urine Na(+) output only on days 3-5, and after 7 days plasma [Na(+)] and [K(+)] were unaffected. In contrast, the CNT-KO mice were highly susceptible to a 2-day 5% K(+) diet and showed lower food intake and relative body weight, lower plasma [Na(+)], higher fractional excretion (FE) of Na(+), higher plasma [K(+)], and lower FE of K(+). The higher FE of Na(+) coincided with lower abundance and phosphorylation of the Na(+)-Cl(-) cotransporter. In conclusion, reducing ENaC expression in the CNT induces clear PHA-1 symptoms during high dietary K(+) loading.

The zinc transporter ZNT3 co-localizes with insulin in INS-1E pancreatic beta cells and influences cell survival, insulin secretion capacity, and ZNT8 expression.

Zinc trafficking in pancreatic beta cells is tightly regulated by zinc transporting (ZNTs) proteins. The role of different ZNTs in the beta cells is currently being clarified. ZNT8 transports zinc into insulin granules and is critical for a correct insulin crystallization and storage in the granules whereas ZNT3 knockout negatively affects beta cell function and survival. Here, we describe for the first time the sub-cellular localization of ZNT3 by immuno-gold electron microscopy and supplement previous data from knockout experiments with investigations of the effect of ZNT3 in a pancreatic beta cell line, INS-1E overexpressing ZNT3. In INS-1E cells, we found that ZNT3 was abundant in insulin containing granules located close to the plasma membrane. The level of ZNT8 mRNA was significantly decreased upon over-expression of ZNT3 at different glucose concentrations (5, 11 and 21 mM glucose). ZNT3 over-expression decreased insulin content and insulin secretion whereas ZNT3 over-expression improved the cell survival after 24 h at varying glucose concentrations (5, 11 and 21 mM). Our data suggest that ZNT3 and ZNT8 (known to regulate insulin secretion) have opposite effects on insulin synthesis and secretion possibly by a transcriptional co-regulation since mRNA expression of ZNT3 was inversely correlated to ZNT8 and ZNT3 over-expression reduced insulin synthesis and secretion in INS-1E cells. ZNT3 over-expression improved cell survival.

Hepatic AQP9 expression in male rats is reduced in response to PPARα agonist treatment.

The peroxisome proliferator receptor α (PPARα) is a key regulator of the hepatic response to fasting with effects on both lipid and carbohydrate metabolism. A role in hepatic glycerol metabolism has also been found; however, the results are somewhat contradictive. Aquaporin 9 (AQP9) is a pore-forming transmembrane protein that facilitates hepatic uptake of glycerol. Its expression is inversely regulated by insulin in male rodents, with increased expression during fasting. Previous results indicate that PPARα plays a crucial role in the induction of AQP9 mRNA during fasting. In the present study, we use PPARα agonists to explore the effect of PPARα activation on hepatic AQP9 expression and on the abundance of enzymes involved in glycerol metabolism using both in vivo and in vitro systems. In male rats with free access to food, treatment with the PPARα agonist WY 14643 (3 mg·kg(-1)·day(-1)) caused a 50% reduction in hepatic AQP9 abundance with the effect being restricted to AQP9 expressed in periportal hepatocytes. The pharmacological activation of PPARα had no effect on the abundance of GlyK, whereas it caused an increased expression of hepatic GPD1, GPAT1, and L-FABP protein. In WIF-B9 and HepG2 hepatocytes, both WY 14643 and another PPARα agonist GW 7647 reduced the abundance of AQP9 protein. In conclusion, pharmacological PPARα activation results in a marked reduction in the abundance of AQP9 in periportal hepatocytes. Together with the effect on the enzymatic apparatus for glycerol metabolism, our results suggest that PPARα activation in the fed state directs glycerol into glycerolipid synthesis rather than into de novo synthesis of glucose.

Coordinated regulation of TRPV5-mediated Ca²âº transport in primary distal convolution cultures.

Fine-tuning of renal calcium ion (Ca(2+)) reabsorption takes place in the distal convoluted and connecting tubules (distal convolution) of the kidney via transcellular Ca(2+) transport, a process controlled by the epithelial Ca(2+) channel Transient Receptor Potential Vanilloid 5 (TRPV5). Studies to delineate the molecular mechanism of transcellular Ca(2+) transport are seriously hampered by the lack of a suitable cell model. The present study describes the establishment and validation of a primary murine cell model of the distal convolution. Viable kidney tubules were isolated from mice expressing enhanced Green Fluorescent Protein (eGFP) under the control of a TRPV5 promoter (pTRPV5-eGFP), using Complex Object Parametric Analyser and Sorting (COPAS) technology. Tubules were grown into tight monolayers on semi-permeable supports. Radioactive (45)Ca(2+) assays showed apical-to-basolateral transport rates of 13.5 ± 1.2 nmol/h/cm(2), which were enhanced by the calciotropic hormones parathyroid hormone and 1,25-dihydroxy vitamin D3. Cell cultures lacking TRPV5, generated by crossbreeding pTRPV5-eGFP with TRPV5 knockout mice (TRPV5(-/-)), showed significantly reduced transepithelial Ca(2+) transport (26 % of control), for the first time directly confirming the key role of TRPV5. Most importantly, using this cell model, a novel molecular player in transepithelial Ca(2+) transport was identified: mRNA analysis revealed that ATP-dependent Ca(2+)-ATPase 4 (PMCA4) instead of PMCA1 was enriched in isolated tubules and downregulated in TRPV5(-/-) material. Immunohistochemical stainings confirmed co-localization of PMCA4 with TRPV5 in the distal convolution. In conclusion, a novel primary cell model with TRPV5-dependent Ca(2+) transport characteristics was successfully established, enabling comprehensive studies of transcellular Ca(2+) transport.

Essential role of the electroneutral Na+-HCO3- cotransporter NBCn1 in murine duodenal acid-base balance and colonic mucus layer build-up in vivo.

Duodenal epithelial cells need efficient defence strategies during gastric acidification of the lumen, while colonic mucosa counteracts damage by pathogens by building up a bacteria-free adherent mucus layer. Transport of HCO3(-) is considered crucial for duodenal defence against acid as well as for mucus release and expansion, but the transport pathways involved are incompletely understood. This study investigated the significance of the electroneutral Na(+)-HCO3(-) cotransporter NBCn1 for duodenal defence against acid and colonic mucus release. NBCn1 was localized to the basolateral membrane of duodenal villous enterocytes and of colonic crypt cells, with predominant expression in goblet cells. Duodenal villous enterocyte intracellular pH was studied before and during a luminal acid load by two-photon microscopy in exteriorized, vascularly perfused, indicator (SNARF-1 AM)-loaded duodenum of isoflurane-anaesthetized, systemic acid-base-controlled mice. Acid-induced HCO3(-) secretion was measured in vivo by single-pass perfusion and pH-stat titration. After a luminal acid load, NBCn1-deficient duodenocytes were unable to recover rapidly from intracellular acidification and could not respond adequately with protective HCO3(-) secretion. In the colon, build-up of the mucus layer was delayed, and a decreased thickness of the adherent mucus layer was observed, suggesting that basolateral HCO3(-) uptake is essential for optimal release of mucus. The electroneutral Na(+)-HCO3(-) cotransporter NBCn1 displays a differential cellular distribution in the murine intestine and is essential for HCO3(-)-dependent mucosal protective functions, such as recovery of intracellular pH and HCO3(-) secretion in the duodenum and secretion of mucus in the colon.

Phosphorylation of aquaporin-2 regulates its endocytosis and protein-protein interactions.

The water channel aquaporin-2 (AQP2) is essential for urine concentration. Vasopressin regulates phosphorylation of AQP2 at four conserved serine residues at the COOH-terminal tail (S256, S261, S264, and S269). We used numerous stably transfected Madin-Darby canine kidney cell models, replacing serine residues with either alanine (A), which prevents phosphorylation, or aspartic acid (D), which mimics the charged state of phosphorylated AQP2, to address whether phosphorylation is involved in regulation of (i) apical plasma membrane abundance of AQP2, (ii) internalization of AQP2, (iii) AQP2 protein-protein interactions, and (iv) degradation of AQP2. Under control conditions, S256D- and 269D-AQP2 mutants had significantly greater apical plasma membrane abundance compared to wild type (WT)-AQP2. Activation of adenylate cyclase significantly increased the apical plasma membrane abundance of all S-A or S-D AQP2 mutants with the exception of 256D-AQP2, although 256A-, 261A-, and 269A-AQP2 mutants increased to a lesser extent than WT-AQP2. Biotin internalization assays and confocal microscopy demonstrated that the internalization of 256D- and 269D-AQP2 from the plasma membrane was slower than WT-AQP2. The slower internalization corresponded with reduced interaction of S256D- and 269D-AQP2 with several proteins involved in endocytosis, including Hsp70, Hsc70, dynamin, and clathrin heavy chain. The mutants with the slowest rate of internalization, 256D- and 269D-AQP2, had a greater protein half-life (t(1/2) = 5.1 h and t(1/2) = 4.4 h, respectively) compared to WT-AQP2 (t(1/2) = 2.9 h). Our results suggest that vasopressin-mediated membrane accumulation of AQP2 can be controlled via regulated exocytosis and endocytosis in a process that is dependent on COOH terminal phosphorylation and subsequent protein-protein interactions.

Absence of E-Cadherin and ß-Catenin in the Basal Plasma Membrane of Collecting Duct Cells During NDI Development and Recovery.

Lithium (Li) induces severe polyuria and polydipsia in up to 40% of patients undergoing Li treatment. In rats, Li treatment induces a reversible cellular remodeling of the collecting duct (CD), decreasing the fraction of principal-to-intercalated cells. To investigate the potential role of adherens junction proteins, we performed immunohistochemistry on kidney cross-sections from rats treated with Li as well as rats undergoing recovery on a normal diet following 4 weeks of Li-treatment. We performed immunoelectron microscopy on cryosections to determine the ultrastructural localizations. Immunohistochemistry showed that E-cadherin and ß-catenin were present in both the lateral and basal plasma membrane domains of CD cells. Immunoelectron microscopy confirmed that ß-catenin was localized both to the lateral and the basal plasma membrane. The basal localization of both proteins was absent from a fraction of mainly principal cells after 10 and 15 days of Li-treatment. After 4 weeks of Li-treatment few to no cells were absent of E-cadherin and ß-catenin at the basal plasma membrane. After 12 and 19 days of recovery some cells exhibited an absence of basal localization of both proteins. Thus, the observed localizational changes of E-cadherin and ß-catenin appear before the cellular remodeling during both development and recovery from Li-NDI.

Genetic ablation of Slc4a10 alters the expression pattern of transporters involved in solute movement in the mouse choroid plexus.

Mutational changes of one transporter can have deleterious effects on epithelial function leaving the cells with the options of either compensating for the loss of function or dedifferentiating. Previous studies have shown that the choroid plexus epithelium (CPE) from mice lacking the Na(+)-dependent Cl(-)/HCO(3)(-) exchanger (NCBE) encoded by Slc4a10 leads to retargeting of the Na(+)/H(+) exchanger 1 (NHE1) from the luminal to the basolateral plasma membrane. We hypothesized that disruption of NCBE, the main basolateral Na(+) importer in the CPE, would lead to a compensatory increase in the abundance of other important transport proteins in this tissue. Aquaporin-1 (AQP1) abundance was 42.7% lower and Na,K-ATPase 36.4% lower in the CPE of Slc4a10 knockout mice, respectively. The NHE1 binding ezrin cytoskeleton appeared disrupted in Slc4a10 knockout mice, whereas no changes were observed in cellular polarization with respect to claudin-2 and appearance of luminal surface microvilli. The renal proximal tubule constitutes a leaky epithelium with high transport rate similar to CPE. Here, Slc4a10 knockout did not affect Na,K-ATPase or AQP1 expression. CPE from AQP1 knockout mice has a secretory defect similar to Slc4a10 mice. However, neither NCBE nor Na,K-ATPase expression was affected in CPE from AQP1 knockout mice. By contrast, the abundance of Na,K-ATPase and NBCe1 was decreased by 23 and 31.7%, respectively, in AQP1 knockout proximal tubules, while the NHE3 abundance was unchanged. In conclusion, CPE lacking NCBE seems to spare the molecular machinery involved in CSF secretion rather than compensate for the loss of the Na(+) loader. Slc4a10 knockout seems to be more deleterious to CPE than AQP1 knockout.