Transepithelial Chloride Transport Research Articles

Cigarette smoke condensate inhibits transepithelial chloride transport and ciliary beat frequency.

Although the pathophysiology leading to rhinosinusitis is complex, evidence indicates that decreased mucociliary clearance (MCC) is a major contributing feature. Normal respiratory epithelial MCC is an important host defense mechanism that is dependent on proper ciliary beating and the biological properties of the airway surface liquid (ASL). The role that tobacco smoke exposure plays as an inhibitor of MCC has yet to be elucidated. The present study investigates the consequences of cigarette smoke exposure on ciliary function and transepithelial chloride (Cl(-)) secretion, a major determinant of ASL. In vitro investigation. Well-characterized primary murine nasal septal epithelial (MNSE) and human sinonasal epithelial (HSNE) cultures were exposed to cigarette smoke condensate (CSC) and compared to control cultures. Effects on ciliary beat frequency (CBF) and Cl(-) secretion were investigated using pharmacologic manipulation. Change in forskolin-stimulated current (DeltaI(SC)), representing transepithelial Cl(-) secretion, was significantly decreased in CSC exposed MNSE (14.97 +/- 1.2 microA/cm(2) vs. control, 19.1 +/- 1.56 microA/cm(2) [P = .04]) and HSNE (2.68 +/- 0.79 muA/cm(2) vs. control, 10.8 +/- 1.73 microA/cm(2)) cultures (P = .001). Forskolin-stimulated CBF was also significantly reduced when acutely exposed to CSC (5.64 +/- 0.06 Hz vs. control 7.15 +/- 0.18 Hz). The present study provides direct evidence that tobacco smoke diminishes two major components of MCC. This links tobacco smoke as a potential contributing and/or exacerbating factor in exposed individuals with chronic rhinosinusitis.

Three Families with Best’s Disease and Normal Electro‐oculogram Recordings

Abstract Purpose To describe 3 families with Best’s disease with normal electro‐oculogram (EOG) and without VMD2 mutations. Methods Evaluation of the patients included visual acuity, fundus and autofluorescence (Heidelberg Retinal Angiograph), Goldmann visual fields, optical coherence tomography (Zeiss, OCT3), full field (ISCEV protocol) and multifocal electroretinograms, and EOG (ISCEV protocol). The diagnosis of Best’s disease was based on autosomal dominant inheritance, typical yellowish, autofluorescent material in the central macula accumulating beneath the retinal pigment epithelium, and decrease of the EOG Arden ratio. Results Among the 1130 families with various retinal dystrophies followed up in Montpellier, 40 (3.5%) were found with vitelliform macular dystrophy. Best’s disease was observed in 20 of them while 13 families had adult macular vitelliform dystrophy and 7 had reticular dystrophy. In the group with Best’s disease, a normal EOG was recorded in 3 families. None of these 3 families carried mutations in VMD2 or RDS, and linkage to the VMD2 locus was excluded in one of them. Conclusion In patients with Best’s disease and VMD2 (bestrophin) mutations, the decreased response of the EOG is attributed to an abnormal transepithelial chloride transport. However, the observation of families with normal EOG and no VMD2 mutations suggests that other genes are responsible for Best’s disease which could not impair the ionic transport but yet lead to similar subretinal deposits.

Barttin binds to the outer lateral surface of the ClC-K2 chloride channel

ClC-K chloride channels belong to the CLC chloride channel family and play an important role in transepithelial chloride transport in the kidney. To be functional, ClC-K channels need to be translocated to the plasma membranes after synthesis; the translocation requires the binding to its β-subunit, barttin. The binding interaction between barttin and ClC-K channels has not been characterized, although the crystal structure of CLC was resolved. In the present study, we sought to clarify the binding sites of barttin in ClC-K2 by co-immunoprecipitation and immunofluorescence microscopy using various ClC-K2 mutants. The deletion of the carboxy-terminal portion of ClC-K2 up to leucine 91, a construct which contains the B domain alone, showed the binding ability to barttin. Since the CLC channel forms an internal antiparallel structure, domain J corresponds to domain B in the carboxy-terminal half of ClC-K. Accordingly, we made the carboxy-terminal half of ClC-K2 containing domain J and thereafter and its deletion mutants, and performed a similar co-immunoprecipitation study. As a result, only domain J was enough for binding to barttin. Immunofluorescence microscopy confirmed that the domains B and J as well as the full length ClC-K2 could be localized to the plasma membranes only when co-expressed with barttin. These results showed that barttin was able to bind to the domains that constitute the outer lateral surfaces of ClC-K2. This information regarding the binding sites will be useful for designing a new class of diuretics or anti-hypertensive agents that inhibit the interaction of ClC-K and barttin.

Role of kidney chloride channels in health and disease

Chloride channels are expressed along the entire mammalian nephron. They participate in transepithelial chloride transport, cell volume regulation and acidification of intracellular vesicles. Some chloride channels are constitutively active and others are regulated by either second messengers such as cAMP or Ca(++) or secondary to changes in membrane potential. The molecular identities of a number of chloride channels within the kidney are still unknown. Abnormalities in chloride channel expression and function in the kidney can cause a range of disorders such as autosomal recessive Dent's disease, Bartter's syndrome, renal tubular acidosis and diabetes insipidus. The purpose of this review is to give an overview of the chloride channels in the kidney and to focus on the function of renal chloride channels as revealed by diseases associated with channel dysfunction.

Chloride transporting capability of Calu‐3 epithelia following persistent knockdown of the cystic fibrosis transmembrane conductance regulator, CFTR

Calu-3 cells are derived from serous cells of human lung submucosal glands, a prime target for therapy in cystic fibrosis (CF). Calu-3 cells can be cultured to form epithelia capable of transepithelial transport of chloride. A CF Calu-3 cell is not available. A retroviral vector was used to cause persistent down regulation of CFTR using siRNA methodology, in Calu-3 cells. A Calu-3 cell line with CFTR content less than 5% of the original line has been established. Epithelia grown using the modified cells have been used in comparative studies of transporting capability. All aspects of cAMP activated chloride secretion were attenuated in the epithelia with reduced CFTR content. However transporting capability was reduced less than the CFTR content. From studies with the CFTR channel inhibitor, GlyH-101, it was concluded that wild type Calu-3 cells have a reserve of CFTR channels not located in the membrane, but available for replacement, while in the modified Calu-3 cell line there was little or no reserve. Lubiprostone, a putative ClC-2 activator, increased transepithelial chloride secretion in both modified and wild type Calu-3 epithelia. Modified Calu-3 epithelia with the residual CFTR currents blocked with GlyH-101 responded equally well to lubiprostone as those without the blocking agent. It appears that lubiprostone is capable of stimulating a non-CFTR dependent transepithelial chloride secretion in Calu-3 monolayers, with obvious implications for CF therapy. Cell lines, however, do not always reflect the behaviour of the native tissue with integrity.

Bioelectric effects of quinine on polarized airway epithelial cells

Quinine has been increasingly utilized as a placebo in cystic fibrosis (CF) clinical trials, including those leading to FDA approval of inhaled tobramycin, recent studies of anti-inflammatory aerosols such as glutathione, and clinical testing of hypertonic saline aerosols to augment mucous clearance. The drug effectively masks taste of experimental therapeutics, but could also confer changes in processes contributing to CF pathogenesis, including chloride secretion and paracellular ion permeability. In the Ussing chamber, concentrations of quinine (1 mg/ml) anticipated in the airways of CF subjects after aerosolization led to changes in chloride transport in Calu-3 (airway serous glandular) cell monolayers. Tissue resistance was significantly disrupted by the compound in both Calu-3 and primary airway epithelial cells in vitro. Lower doses of quinine (between 10 and 100 μg/ml) strongly inhibited the chloride secretory mechanism that utilizes CFTR, and forskolin activated I SC was reduced by approximately 24% and 44% in the presence of 10 and 100 μg/ml quinine, respectively. Our findings indicate that quinine disrupts airway epithelial functional integrity and blocks transepithelial chloride transport. The use of quinine as a taste-masking agent may have bioelectric effects relevant to CF trials using aerosolized drug delivery.

Tissue and subcellular distribution of CLIC1

BackgroundCLIC1 is a chloride channel whose cellular role remains uncertain. The distribution of CLIC1 in normal tissues is largely unknown and conflicting data have been reported regarding the cellular membrane fraction in which CLIC1 resides.ResultsNew antisera to CLIC1 were generated and were found to be sensitive and specific for detecting this protein. These antisera were used to investigate the distribution of CLIC1 in mouse tissue sections and three cultured cell lines. We find CLIC1 is expressed in the apical domains of several simple columnar epithelia including glandular stomach, small intestine, colon, bile ducts, pancreatic ducts, airway, and the tail of the epididymis, in addition to the previously reported renal proximal tubule. CLIC1 is expressed in a non-polarized distribution in the basal epithelial cell layer of the stratified squamous epithelium of the upper gastrointesitinal tract and the basal cells of the epididymis, and is present diffusely in skeletal muscle. Distribution of CLIC1 was examined in Panc1 cells, a relatively undifferentiated, non-polarized human cell line derived from pancreatic cancer, and T84 cells, a human colon cancer cell line which can form a polarized epithelium that is capable of regulated chloride transport. Digitonin extraction was used to distinguish membrane-inserted CLIC1 from the soluble cytoplasmic form of the protein. We find that digitonin-resistant CLIC1 is primarily present in the plasma membrane of Panc1 cells. In T84 cells, we find digitonin-resistant CLIC1 is present in an intracellular compartment which is concentrated immediately below the apical plasma membrane and the extent of apical polarization is enhanced with forskolin, which activates transepithelial chloride transport and apical membrane traffic in these cells. The sub-apical CLIC1 compartment was further characterized in a well-differentiated mouse renal proximal tubule cell line. The distribution of CLIC1 was found to overlap that of megalin and the sodium-phosphate cotransporter, NaPi-II, which are markers of the apical endocytic/recycling compartment in proximal tubule.ConclusionThe cell and tissue specific patterns of CLIC1 expression suggest it may play distinct roles in different cell types. In certain polarized columnar epithelia, it may play a role in apical membrane recycling.

Angiotensin II increases chloride absorption in the cortical collecting duct in mice through a pendrin-dependent mechanism

Pendrin (Slc26a4) localizes to type B and non-A, non-B intercalated cells in the distal convoluted tubule, the connecting tubule, and the cortical collecting duct (CCD), where it mediates apical Cl(-)/HCO(3)(-) exchange. The purpose of this study was to determine whether angiotensin II increases transepithelial net chloride transport, J(Cl), in mouse CCD through a pendrin-dependent mechanism. J(Cl) and transepithelial voltage, V(T), were measured in CCDs perfused in vitro from wild-type and Slc26a4 null mice ingesting a NaCl-replete diet or a NaCl-replete diet and furosemide. In CCDs from wild-type mice ingesting a NaCl-replete diet, V(T) and J(Cl) were not different from zero either in the presence or absence of angiotensin II (10(-8) M) in the bath. Thus further experiments employed mice given the high-NaCl diet and furosemide to upregulate renal pendrin expression. CCDs from furosemide-treated wild-type mice had a lumen-negative V(T) and absorbed Cl(-). With angiotensin II in the bath, Cl(-) absorption doubled although V(T) did not become more lumen negative. In contrast, in CCDs from furosemide-treated Slc26a4 null mice, Cl(-) secretion and a V(T) of approximately 0 were observed, neither of which changed with angiotensin II application. Inhibiting ENaC with benzamil abolished V(T) although J(Cl) fell only approximately 50%. Thus substantial Cl(-) absorption is observed in the absence of an electromotive force. Attenuating apical anion exchange with the peritubular application of the H(+)-ATPase inhibitor bafilomycin abolished benzamil-insensitive Cl(-) absorption. In conclusion, angiotensin II increases transcellular Cl(-) absorption in the CCD through a pendrin- and H(+)-ATPase-dependent process.

Effect of cAMP on Porcine Ciliary Transepithelial Short-Circuit Current, Sodium Transport, and Chloride Transport

To investigate the effects of 8-bromo-cAMP (cAMP) on porcine ciliary transepithelial short-circuit current (Isc) and transport of chloride (Cl-) and sodium (Na+). With Ussing-type chambers, cAMP-induced changes in Isc, electrical resistance (ER), and transepithelial 36Cl- and 22Na+ fluxes were measured. Drugs were applied to the nonpigmented epithelium (NPE) and/or pigmented epithelium (PE) side(s). The effect of IBMX (1, 5, or 10 microM; 3-isobutyl-1-methylxanthine) on Isc-increase induced by 8-bromo-cAMP on the PE side was also tested. On the NPE side, a single concentration (10 microM, 100 microM, or 1 mM) of 8-bromo-cAMP induced a biphasic (transient peak followed by sustained plateau) Isc increase. On the PE side, 8-bromo-cAMP induced a similar but delayed biphasic Isc increase at 1 mM, a slight plateau-Isc increase at 100 microM, and no Isc increase at 10 microM. In the concentration-response curve, the cAMP-induced peak-Isc increase became significant at a concentration 10,000 times lower on the NPE than on the PE side. At 10 microM, the cumulative cAMP-induced Isc-increase reached its maximum on the NPE side, but was virtually nonexistent on the PE side. IBMX (a phosphodiesterase inhibitor) but not 8-CPT-6-Phe-cAMP (higher permeability than 8-bromo-cAMP) significantly increased the peak-Isc concentration-response curve induced by 8-bromo-cAMP (10 nM-1 mM) on the PE side. On the NPE but not the PE side, 10 microM 8-bromo-cAMP induced a significant but transient increase in net PE-to-NPE 36Cl- flux (1.03 +/- 0.18 microEq/min per square centimeter; P < 0.001). Neither ER nor transepithelial 22Na+ flux was changed after cAMP exposure. In porcine ciliary processes, apparently on the NPE side, cAMP triggers a biphasic (transient peak followed by a sustained plateau) Isc increase. Only the peak-Isc increase involves an increase in net PE-to-NPE Cl- transport.

Diagnostik bei chronischem Husten in der Pädiatrie

Coughing is the most frequent symptom which prompts children with their families to consult a physician. Coughing lasting longer than 3 4 weeks requires a careful differential diagnosis. Lung sounds and accompanying symptoms such as atopic markers of the skin, stridor, wheezing, rales, dyspnea, pain, consistency and color of the mucus, mouth breathing during the night, dystrophia etc. are important signs that necessitate the initiation of a rational and adequate diagnostic procedure. The scope of laboratory examinations depends on severity and duration of the disease and comprises the patients history, various lung function tests and, if necessary, invasive procedures. During the last years, new non-invasive methods have been developed which support the diagnosis of cystic fibrosis (electrophysiologic examination at the nasal mucosa to evaluate the transepithelial chloride transport, complete mutation analysis of the cystic fibrosis gene), primary ciliary dyskinesia (measuring nasal (NO)) and obstructive sleep apnea (polysomnography). Modern lung function tests for infants based on improved baby plethysmography allow to separate obstructive from restrictive impairments of the lung in a more reliable manner than before.

Feedback-mediated dynamics in two coupled nephrons

Previously, we developed a dynamic model for the tubuloglomerular feedback (TGF) system in a single, short-looped nephron of the mammalian kidney. In that model, a semi-linear hyperbolic partial differential equation was used to represent two fundamental processes of solute transport in the nephron’s thick ascending limb (TAL): chloride advection by fluid flow along the TAL lumen and transepithelial chloride transport from the lumen to the interstitium. An empirical function and a time delay were used to relate glomerular filtration rate to the chloride concentration at the macula densa of the TAL. Analysis of the model equations indicated that stable limit-cycle oscillations (LCO) in nephron fluid flow and chloride concentration can emerge for sufficiently large feedback gain magnitude and time delay. In this study, the single-nephron model was extended to two nephrons, which were coupled through their filtration rates. Explicit analytical conditions were obtained for bifurcation loci corresponding to two special cases: (1) identical time delays but differing feedback gains, and (2) identical gains but differing delays. Similar to the case of a single nephron, our analysis indicates that stable LCO can emerge in coupled nephrons for sufficiently large gains and delays. However, these LCO may emerge at lower values of the feedback gain, relative to a single (i.e., uncoupled) nephron, or at shorter delays, provided the delays are sufficiently close. These results suggest that, in vivo, if two nephrons are sufficiently similar, then coupling will tend to increase the likelihood of LCO.

110. Helper-Dependent Adenovirus with the Cytokeratin-18 Promoter Produces Greater Transepithelial Chloride Current and More Persistent Transgene Expression Than the CMV Promoter in Differentiated Human Airway Epithelia

Gene transfer of the wild-type CFTR cDNA to airway epithelia could rescue the electrolyte transport defect and represent an attractive strategy for treating the genetic disease cystic fibrosis (CF). Numerous gene transfer vectors have expressed CFTR under control of the cytomegalovirus (CMV) enhancer/promoter because it generates robust expression in many cell types. However, this promoter could drive expression in cell types that do not normally express CFTR and may yield limited persistence. An alternative enhancer/promoter is from the cytoskeletal protein cytokeratin-18 (K18); cytokeratin-18 is constitutively expressed in differentiated epithelia and has a tissue distribution similar to CFTR. To test the K18 promoter, we used helper-dependent adenoviral vectors and compared transgene expression driven by the K18 and CMV promoters. We delivered them to well-differentiated primary cultures of CF airway epithelia grown at the air-liquid interface. We applied vector to the basolateral surface to facilitate infection through binding of the adenovirus receptor CAR. With a β-galactosidase transgene, the CMV promoter generated much greater β-galactosidase activity and total protein levels than the K18 promoter. However, the opposite pattern emerged when the two promoters drove CFTR expression; the K18 promoter generated greater transepithelial chloride current than the CMV promoter. These results suggested that the two promoters might drive expression in different cell types. We found that the CMV promoter generated the most intense X-Gal staining in basal cells. In contrast, the K18 promoter generated diffuse staining in columnar epithelial cells. These results suggest that the K18 promoter produced relatively greater CFTR expression in columnar epithelial cells that touch the apical surface and are responsible for transepithelial chloride transport, whereas the CMV promoter generated greater expression in basal cells. We also compared the persistence of transgene expression. Over the course of 12 weeks, transgene expression driven by the K18 promoter remained relatively constant, while transgene expression from the CMV promoter fell rapidly. These data emphasize the importance of appropriate regulatory elements in developing gene transfer vectors for CF airway epithelia and suggest that the K18 promoter may have advantages over the CMV promoter.

A common sequence variation of the CLCNKB gene strongly activates ClC-Kb chloride channel activity

Tubular transepithelial reabsorption of chloride along the nephron is a major determinant of body salt and water homeostasis and blood pressure regulation. About 40% of the glomerulary filtered sodium chloride are reabsorbed in the distal nephrons. Vectorial transepithelial sodium chloride transport is critically dependent on the function of basolateral ClC-K type chloride channels there. Modulation of ClC-Kb chloride channel activity by polymorphic variations of the CLCNKB gene, thus, could form a molecular basis for salt sensitivity of blood pressure regulation. In this study we tested the effect of several polymorphic variants on ClC-Kb chloride channel activity. After heterologous expression in Xenopus oocytes, ClC-Kb channel activity and surface expression in presence of the ClC-K beta subunit barttin were determined by two-electrode voltage-clamp analysis, immunofluorescence, and ClC-Kb surface enzyme-linked immunosorbent assay (ELISA). Chloride currents induced by the ClC-Kb variants L27R, G214A, I419V, T562M, and E578K were not significantly different from wild-type currents. The ClC-KbT481S variation, however, which showed a frequency of 20% in our control population, dramatically activated chloride conductance by a factor of 20. Activation of chloride currents was also observed after introducing homologous mutations in ClC-Ka and ClC-K1, but not in ClC-2 and ClC-5 chloride channels. ClC-Kb activation by the T481S mutation did not change intrinsic ion channel pore properties and did not require increased surface expression of ClC-KbT481S. Genetic heterogeneity of ClC-Kb chloride channels correlates with functional heterogeneity, which assigns ClC-Kb to a set of genes potentially relevant for polygenic salt-sensitivity of blood pressure regulation.

A Multicenter Study of the Effect of Solution Temperature on Nasal Potential Difference Measurements

Nasal potential difference (PD) measurement quantifies the abnormal sodium and chloride transport that is characteristic of cystic fibrosis (CF) and has gained acceptance as both a diagnostic tool and outcome measure for new CF therapies. Because small changes in nasal PD-measured chloride transport are often an important component in evaluating new CF therapies, techniques to maximize sensitivity and reproducibility are essential. To determine if administration of warmed nasal PD solutions (37 degrees C), instead of room temperature solutions (22 degrees C), results in significant increase in nasal PD-measured transepithelial chloride transport. Multicenter, prospective, cross-over trial of repeated measurements of nasal PD at 22 degrees C and 37 degrees C. Thirty-two healthy volunteers completed the study (four centers, each with 8 subjects). For 22 degrees C vs 37 degrees C, baseline (+/- SD) nasal PD (- 19.3 +/- 6.9 millivolts [mV] vs - 18.8 +/- 7.7 mV, p = 0.76), amiloride-sensitive PD (Delta PD, 10.4 +/- 5.6 mV vs 11.0 +/- 6.1 mV, p = 0.60), and low chloride response (Delta PD, - 10.0 +/- 8.0 mV vs - 8.0 +/- 7.1 mV, p = 0.13) were not statistically significantly affected by warming of solutions. Warming solutions to 37 degrees C dramatically increased the chloride transport response to isoproterenol (Delta PD, - 6.9 +/- 6.4 mV vs - 13.3 +/- 8.8 mV, p < 0.01) and the combined total response to low chloride and isoproterenol (Delta PD, - 16.9 +/- 9.5 mV vs - 21.3 +/- 11.9 mV, p = 0.01). The average increases observed with warming in isoproterenol and combined total responses were - 6.4 mV (95% confidence interval [CI], - 8.5 to - 4.3) and - 4.4 mV (95% CI, - 7.6 to - 1.1), respectively. Performing nasal PD studies with solutions at 37 degrees C instead of 22 degrees C increases the observed total chloride response by approximately 25% and the isoproterenol-dependent chloride response by approximately 95%. The Cystic Fibrosis Foundation Therapeutics Development Network now recommends warming of solutions as a standard procedure for nasal PD protocols. Utilization of warmed solutions will standardize nasal PD techniques across centers and potentially increase the ability to identify therapies that result in small incremental improvements in CF transmembrane conductance regulator function.

Large Chloride Channel from Pre-eclamptic Human Placenta

Chloride transport involving conductive pathways participates in numerous epithelial functions, such as membrane voltage maintenance, solute transport and cell volume regulation. Evidence points to involvement of transepithelial chloride transport in such functions in placental syncytiotrophoblast. A molecular candidate for physiologic conductive chloride transport in apical syncytiotrophoblast membrane is a Maxi-chloride channel with distinct biophysical properties: conductance over 200 pS, multiple substates, voltage dependent open probability, and permeation to anionic amino acids. Pre-eclampsia, a high incidence pathology of pregnancy, exerts great impact on fetal morbi-mortality. This relies, among others, on intrauterine growth restriction (IUGR), thought to be mediated by diminished blood flow to the placenta, with growing knowledge regarding contribution of other factors. The Maxi-chloride channel's properties suggest it could be altered in this pathology. We have characterized the apical chloride channels from pre-eclamptic placentae, reconstituted in giant liposomes suitable for patch-clamp electrophysiological studies. In n=33 experiments from n=6 pre-eclamptic placentae we observed a chloride-permeable channel with similar biophysical properties to the channel from normal tissue ( n=29 experiments from n=15 placentae). However, the main conductance state showed diminished magnitude (<150 pS), and the open probability versus voltage relationship exhibited a flattened curve instead of the bell-shaped curve of normal placentae. These results are the first evidence of a functionally altered ionic channel from placental syncytiotrophoblast in pre-eclampsia. Considering the abundance of chloride-conducting channel activity in human apical membrane and their relevance in epithelial function in general, these alterations could greatly disturb numerous placental functions that rely on syncytiotrophoblast integrity.

Detection of ClC-3 and ClC-5 in epididymal epithelium: immunofluorescence and RT-PCR after LCM.

Epithelial cells of the epididymis and vas deferens establish an optimum luminal environment in which spermatozoa mature and are stored. This is achieved by active transepithelial transport of various ions including Cl(-) and H(+). We investigated the localization of three closely related members of the ClC family, ClC-3, ClC-4, and ClC-5, in the epididymis and vas deferens. RT-PCR using mRNA isolated by laser capture microdissection (LCM)-detected ClC-3 and ClC-5 transcripts but did not detect any ClC-4-specific transcript. Western blot and immunofluorescence analysis demonstrated that ClC-3 and ClC-5 proteins are present in all regions of the epididymis and in the vas deferens. ClC-5 is expressed exclusively in H(+)-ATPase-rich cells (narrow and clear cells). Confocal microscopy showed that ClC-5 partially colocalizes with the H(+)-ATPase in the subapical pole of clear cells. ClC-3 is strongly expressed in the apical membrane of principal cells of the caput epididymidis and the vas deferens and is less abundant in principal cells of the body and cauda epididymidis. These findings are consistent with a potential role for ClC-3 in transepithelial chloride transport by principal cells and for ClC-5 in the acidification of H(+)-ATPase-containing vesicles in narrow and clear cells. ClC-5 might facilitate endosome trafficking in the epididymis, as has been proposed in the kidney.

Renal and Intestinal Handling of Oxalate following Oxalate Loading in Rats

Background: The enteric excretion of oxalate has been established in rats with chronic renal failure induced by 5/6 nephrectomy [Hatch et al.: Regulatory aspects of oxalate secretion in enteric oxalate elimination. JASN 1999;10:S324] and this response is mediated by angiotensin II receptor activation. However, the renal and intestinal handling of oxalate has not been evaluated for other common models of hyperoxaluria that simulate primary hyperoxaluria or oxalate stone disease. Methods: We assessed the renal clearances of creatinine, oxalate and calcium in three rat models: chronic hyperoxaluria (CH), chronic hyperoxaluria with hyperoxalemia (CHH) and acute hyperoxaluria (AH), and evaluated the transepithelial transport of oxalate and chloride in large intestinal segments of these models and their sensitivity to angiotensin II antagonism. Results: Hyperoxaluria alone (CH) was not associated with changes in colonic oxalate transport, whereas changes in net oxalate transport in distal colon from absorption to net secretion was observed in models with hyperoxalemia (CHH and AH). Angiotensin II receptor antagonism with losartan reduced net colonic oxalate secretion in AH but not CHH. Conclusions: Colonic secretion of oxalate is stimulated in rat models exhibiting hyperoxalemia suggesting a contribution of this extrarenal pathway to regulation of oxalate mass balance in clinical conditions manifesting hyperoxalemia. The transport avenues and regulatory mechanisms may not be identical to those observed during adaptive enteric oxalate secretion in chronic renal failure models.

Alkalinization by chloride/bicarbonate pathway in larval mosquito midgut.

The midgut of mosquito larvae maintains a specific lumen alkalinization profile with large longitudinal gradients (pH approximately 3 units*mm(-1)) in which an extremely alkaline (pH approximately 11) anterior midgut lies between near-neutral posterior midgut and gastric cecum (pH 7-8). A plasma membrane H(+) V-ATPase energizes this alkalinization but the ion carriers involved are unknown. Capillary zone electrophoresis of body samples with outlet conductivity detection showed a specific transepithelial distribution of chloride and bicarbonate/carbonate ions, with high concentrations of both anions in the midgut tissue: 68.3 +/- 5.64 and 50.8 +/- 4.21 mM, respectively. Chloride was higher in the hemolymph, 57.6 +/- 7.84, than in the lumen, 3.51 +/- 2.58, whereas bicarbonate was higher in the lumen, 58.1 +/- 7.34, than the hemolymph, 3.96 +/- 2.89. Time-lapse video assays of pH profiles in vivo revealed that ingestion of the carbonic anhydrase inhibitor acetazolamide and the ion exchange inhibitor DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid), at 10(-4) M eliminates lumen alkalinization. Basal application of these inhibitors in situ also reduced gradients recorded with self-referencing pH-sensitive microelectrodes near the basal membrane by approximately 65% and 85% respectively. Self-referencing chloride-selective microelectrodes revealed a specific spatial profile of transepithelial chloride transport with an efflux maximum in anterior midgut. Both acetazolamide and DIDS reduced chloride effluxes. These data suggest that an H(+) V-ATPase-energized anion exchange occurs across the apical membrane of the epithelial cells and implicate an electrophoretic Cl(-)/HCO(3)(-) exchanger and carbonic anhydrase as crucial components of the steady-state alkalinization in anterior midgut of mosquito larvae.

Inhibition by mercuric chloride of Na-K-2Cl cotransport activity in rectal gland plasma membrane vesicles isolated from Squalus acanthias

The rectal gland of the dogfish shark is a model system for active transepithelial transport of chloride. It has been shown previously that mercuric chloride, one of the toxic environmental pollutants, inhibits chloride secretion in this organ. In order to investigate the mechanism of action of HgCl2 at a membrane-molecular level, plasma membrane vesicles were isolated from the rectal gland and the effect of mercury on the activity of the Na-K-2Cl cotransporter was investigated in isotope flux studies. During a 30 s exposure HgCl2 inhibited cotransport activity in a dose-dependent manner with an apparent Ki of approx. 50 μM. The inhibition was complete after 15 s, partly reversible by dilution of the incubation medium and completely attenuated upon addition of reduced glutathione. The extent of inhibition by mercury depended on the ionic composition of the medium. The sensitivity of the cotransporter was highest when only the high affinity binding sites for sodium and chloride were saturated. Organic mercurials such as p-chloromercuribenzoic acid and p-chloromercuriphenylsulfonic acid at 100 μM did not inhibit the cotransporter, similarly exposure of the vesicles to 10 mM H2O2 or 1 mM dithiothreitol for 30 min at 15°C did not change cotransport activity. Transport activity was, however, reduced by 45.9±2.5% after an incubation with 3 mM N-ethylmaleimide for 20 min. Blocking free amino groups by N-hydroxysuccinimide or biotinamidocapronate-N-hydroxysulfosuccinimide had no effect. Investigations on the sidedness of the plasma membrane vesicles, employing the asymmetry of the (Na+K)-ATPase, demonstrated a right-side-out orientation in which the former extracellular face of the membrane is exposed to the incubation medium. In addition, extracellular mercury (5×10−5 M) inhibited bumetanide-sensitive rubidium uptake into T84 cells by 48.5±7.1% after a 2 min incubation period. This inhibition was reversible in a manner similar to that observed in the plasma membrane vesicles. These studies suggest that in isolated rectal gland plasma membrane vesicles the Na-K-2Cl cotransporter (sNKCC1) exposes functionally relevant mercury binding sites at its external surface. These sites represent probably cysteines, the accessibility and/or sensitivity of which depends on the functional state of the transporter.

Physiological role of CLC-K1 chloride channel in the kidney.

Although CLC chloride channels share a common structure, the sites of expression in the body and their intracellular localization are different among CLCs. CLC-K1 and -K2 are kidney-specific CLC chloride channels. We have clarified their localization in the plasma membranes of tubular cells by immunohistochemistry and proposed their roles in transepithelial chloride transport. Since there exit no good inhibitors for these channels, a gene knockout approach was the only way to establish their roles in kidney. While we were generating CLC-K knockout mice, Simon et al. reported that the mutations of CLC-K2 in human resulted in Bartter's syndrome. This had been anticipated since CLC-K2 is known to be present in the basolateral plasma membranes of the distal nephron where sodium-dependent chloride transporters are present in the apical membranes. Thus, CLC-K2 constitutes an important route for chloride reabsorption as an exit for chloride ions in the basolateral membrane. Another important finding in Simon's report was that no CLC-K (a human homologue of rat CLC-K1) mutation was found in patients with Bartter's syndrome. This suggested that CLC-K1 has a different role in kidney. We believed this to be true, based on the finding that the intrarenal localization of CLC-K1 and CLC-K2 are completely different. In the CLC-K1 knockout mice, we could clearly verify that (i) the high chloride permeability in the tAL was mediated by CLC-K1 and (ii) this chloride transport is necessary for urinary concentration. Further studies are necessary to elucidate the detailed mechanisms of the urinary-concentrating defect in Clcnk1 -/- mice. Exact clearance studies and measurements of osmolality and solute contents in the inner medulla will provide the answer to this question.