Na(+)/H(+) exchanger 3 (NHE3) is expressed in the brush border (BB) of intestinal epithelial cells and accounts for the majority of neutral NaCl absorption. It has been shown that the Na(+)/H(+) exchanger regulatory factor (NHERF) family members of multi-PDZ domain-containing scaffold proteins bind to the NHE3 COOH terminus and play necessary roles in NHE3 regulation in intestinal epithelial cells. Most studies of NHE3 regulation have been in cell models in which NHERF1 and/or NHERF2 were overexpressed. We have now developed an intestinal Na(+) absorptive cell model in Caco-2/bbe cells by expressing hemagglutinin (HA)-tagged NHE3 with an adenoviral infection system. Roles of NHERF1 and NHERF2 in NHE3 regulation were determined, including inhibition by cAMP, cGMP, and Ca(2+) and stimulation by EGF, with knockdown (KD) approaches with lentivirus (Lenti)-short hairpin RNA (shRNA) and/or adenovirus (Adeno)-small interfering RNA (siRNA). Stable infection of Caco-2/bbe cells by NHERF1 or NHERF2 Lenti-shRNA significantly and specifically reduced NHERF protein expression by >80%. NHERF1 KD reduced basal NHE3 activity, while NHERF2 KD stimulated NHE3 activity. siRNA-mediated (transient) and Lenti-shRNA-mediated (stable) gene silencing of NHERF2 (but not of NHERF1) abolished cGMP- and Ca(2+)-dependent inhibition of NHE3. KD of NHERF1 or NHERF2 alone had no effect on cAMP inhibition of NHE3, but KD of both simultaneously abolished the effect of cAMP. The stimulatory effect of EGF on NHE3 was eliminated in NHERF1-KD but occurred normally in NHERF2-KD cells. These findings show that both NHERF2 and NHERF1 are involved in setting NHE3 activity. NHERF2 is necessary for cGMP-dependent protein kinase (cGK) II- and Ca(2+)-dependent inhibition of NHE3. cAMP-dependent inhibition of NHE3 activity requires either NHERF1 or NHERF2. Stimulation of NHE3 activity by EGF is NHERF1 dependent.

ClC-5, a chloride/proton exchanger, is predominantly expressed and localized in subapical endosomes of the renal proximal tubule. Mutations of the CLCN5 gene cause Dent disease. The symptoms of Dent disease are replicated in Clcn5 knockout mice. Absence of ClC-5 in mice is associated with reduced surface expression of NHE3 in proximal tubules. The molecular basis for this change is not fully understood. In this study, we investigated the mechanisms by which ClC-5 regulates trafficking of NHE3. Whether ClC-5-dependent endocytosis, exocytosis, or both contributed to the altered distribution of NHE3 was examined. First, NHE3 activity in proximal tubules of wild type (WT) and Clcn5 KO mice was determined by two-photon microscopy. Basal and dexamethasone-stimulated NHE3 activity of Clcn5 KO mice was decreased compared with that seen in WT mice, whereas the degree of inhibition of NHE3 activity by increasing cellular concentration of cAMP (forskolin) or Ca2+ (A23187) was not different in WT and Clcn5 KO mice. Second, NHE3-dependent absorption of HCO3-, measured by single tubule perfusion, was reduced in proximal tubules of Clcn5 KO mice. Third, by cell surface biotinylation, trafficking of NHE3 was examined in short hairpin RNA (shRNA) plasmid-transfected opossum kidney cells. Surface NHE3 was reduced in opossum kidney cells with reduced expression of ClC-5, whereas the total protein level of NHE3 did not change. Parathyroid hormone decreased NHE3 surface expression, but the extent of decrease and the rate of endocytosis observed in both scrambled and ClC-5 knockdown cells were not significantly different. However, the rates of basal and dexamethasone-stimulated exocytosis of NHE3 were attenuated in ClC-5 knockdown cells. These results show that ClC-5 plays an essential role in exocytosis of NHE3.

A processing technique based on the principle of phase separation was developed to fabricate three-dimensional microcellular foams to act as templates for cell transplantation. The polymers used to make the foams were polylactic acid (PLLA) and a polyphosphoester (BPA/PP). The resulting foams had relatively uniform, open cells throughout the matrix. The foams could also be fabricated into complex shapes to meet specific design requirements. The foam morphology and microstructure were characterized by mercury porosimetry and scanning electron microscopy. Osteoblast like cells ROS17/2.8 were successfully cultured in the foams. Cell attachment to the foam interior was verified by confocal microscopy. The fabrication technique allows incorporation of drugs or nutrients into the highly porous structure as demonstrated by the intimate dispersion of fluorescein isothiocyanate (FITC) in the matrix.

Voltage-dependent L-type calcium channels (VDCC) play important roles in many cellular processes. The interaction of the actin cytoskeleton with the channel in nonexcitable cells is less well understood. We performed whole-cell patch-clamp surface biotinylation and calcium imaging on different osteoblast cells to determine channel kinetics, amplitude, surface abundance, and intracellular calcium, respectively. Patch-clamp studies showed that actin polymerization by phalloidin increased the peak current density of I Ca , whereas actin depolymerization by cytochalasin D (CD) significantly decreased the current amplitude. This result is consistent with calcium imaging, which showed that CD significantly decreased Bay K8644-induced intracellular calcium increase. Surface biotinylation studies showed that CD is not able to affect the surface expression of the pore-forming subunit α 1C . Interestingly, application of CD caused a significantly negative shift in the steady-state inactivation kinetics of I Ca . There were decreases in the voltage at half-maximal inactivation that changed in a dose-dependent manner. CD also reduced the effect of activated vitamin D 3 (1α,25-D3) on VDCC and intracellular calcium. We conclude that in osteoblasts the actin cytoskeleton affects α 1C by altering the channel kinetic properties, instead of changing the surface expression, and it is able to regulate 1α,25-D3 signaling through VDCC. Our study provides a new insight into calcium regulation in osteoblasts, which are essential in many physiological functions of this cell.

Although the intracellular Cl−/H+ exchanger Clc-5 is expressed in apical intestinal endocytic compartments, its pathophysiological role in the gastrointestinal tract is unknown. In light of recent findings that CLC-5 is downregulated in active ulcerative colitis (UC), we tested the hypothesis that loss of CLC-5 modulates the immune response, thereby inducing susceptibility to UC. Acute dextran sulfate sodium (DSS) colitis was induced in Clcn5 knockout (KO) and wild-type (WT) mice. Colitis, monitored by disease activity index, histological activity index, and myeloperoxidase activity were significantly elevated in DSS-induced Clcn5 KO mice compared with those in WT mice. Comprehensive serum multiplex cytokine profiling demonstrated a heightened Th1–Th17 profile (increased TNF-α, IL-6, and IL-17) in DSS-induced Clcn5 KO mice compared with that in WT DSS colitis mice. Interestingly, Clcn5 KO mice maintained on a high vitamin D diet attenuated DSS-induced colitis. Immunofluorescence and Western blot analyses of colonic mucosa validated the systemic cytokine patterns and further revealed enhanced activation of the NF-κB pathway in DSS-induced Clcn5 KO mice compared with those in WT mice. Intriguingly, high baseline levels of IL-6 and phospho-IκB were observed in Clcn5 KO mice, suggesting a novel immunopathogenic role for the functional defects that result from the loss of Clc-5. Our studies demonstrate that the loss of Clc-5 1) exhibits IL-6–mediated immunopathogenesis, 2) significantly exacerbated DSS-induced colitis, which is influenced by dietary factors, including vitamin D, and 3) portrays distinct NF-κB–modulated Th1–Th17 immune dysregulation, implying a role for CLC-5 in the immunopathogenesis of UC.

The cystic fibrosis transmembrane regulator (CFTR) is the major Cl channel found in the apical membrane of intestinal epithelial cells. Protein kinase A (PKA) phosphorylates and opens CFTR. E xchange p rotein directly a ctivated by c AMP (Epac) upon binding of cAMP, activates PKA‐independent signaling via small G proteins. We tested the hypothesis that Epac are involved in cAMP‐stimulated Cl secretion. Human intestinal T84 cells and mouse intestine were used for short circuit current (Isc) measurement in response to agonist stimulated Cl secretion. Epac activator, 8‐pCPT‐2′‐O‐Me‐cAMP, elevated [Ca 2+ ]i, activated Rap2 protein and induced Cl secretion in intact and basolateral membrane permeabilized T84 cells and mouse ileum. These effects of 8‐pCPT‐2′‐O‐Me‐cAMP were completely abolished by BAPTA‐AM, but not by H89. In contrast, T84 cells with silenced Epac1 protein had reduced Isc response to forskolin which was completely inhibited by H89 but not by the phospholipase C inhibitor, U73122. The lack of effect of 8‐pCPT‐2′‐O‐Me‐cAMP either to CFTRinhi‐172, glibenclamide or on whole cell patch clamp recording of Cl current in CHO cells transiently expressing the human CFTR, implicates that CFTR channels are not involved. Biophysical characterization of the Epac1‐dependent Cl conductance in Ussing chambers suggests that Epac mediated Cl conductance was hyperpolarization‐activated, inward rectifying, and displays a Cl − >Br − >I − permeability sequence. These results lead us to conclude that the Epac‐Rap‐PLC‐[Ca 2+ ]i signaling pathway is involved in cAMP‐stimulated Cl secretion through non‐CFTR Cl channel.

Intestinal Cl- secretion is stimulated by cyclic AMP (cAMP) and intracellular calcium ([Ca2+]i). Recent studies show that protein kinase A (PKA) and the exchange protein directly activated by cAMP (Epac) are downstream targets of cAMP. Therefore, we tested whether both PKA and Epac are involved in forskolin (FSK)/cAMP-stimulated Cl- secretion. Human intestinal T84 cells and mouse small intestine were used for short circuit current (I(sc)) measurement in response to agonist-stimulated Cl- secretion. FSK-stimulated Cl- secretion was completely inhibited by the additive effects of the PKA inhibitor, H89 (1 microM), and the [Ca2+]i chelator, 1,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM; 25 microM). Both FSK and the Epac activator 8-pCPT-2'-O-Me-cAMP (50 microM) elevated [Ca2+]i, activated Ras-related protein 2, and induced Cl- secretion in intact or basolateral membrane-permeabilized T84 cells and mouse ileal sheets. The effects of 8-pCPT-2'-O-Me-cAMP were completely abolished by BAPTA-AM, but not by H89. In contrast, T84 cells with silenced Epac1 had a reduced I(sc) response to FSK, and this response was completely inhibited by H89, but not by the phospholipase C inhibitor U73122 or BAPTA-AM. The stimulatory effect of 8-pCPT-2'-O-Me-cAMP on Cl- secretion was not abolished by cystic fibrosis transmembrane conductance (CFTR) inhibitor 172 or glibenclamide, suggesting that CFTR channels are not involved. This was confirmed by lack of effect of 8-pCPT-2'-O-Me-cAMP on whole cell patch clamp recordings of CFTR currents in Chinese hamster ovary cells transiently expressing the human CFTR channel. Furthermore, biophysical characterization of the Epac1-dependent Cl- conductance of T84 cells mounted in Ussing chambers suggested that this conductance was hyperpolarization activated, inwardly rectifying, and displayed a Cl->Br->I- permeability sequence. These results led us to conclude that the Epac-Rap-PLC-[Ca2+]i signaling pathway is involved in cAMP-stimulated Cl- secretion, which is carried by a novel, previously undescribed Cl- channel.

This study investigated whether repeated administration of recombinant adeno-associated virus type 5 (rAAV5) to the airways induces inflammatory processes in the lungs of BALB/c-mice, with mechanical and histologic changes. Saline was instilled intratracheally in the control group, and rAAV5-green fluorescence protein (GFP) (4 × 1011 particles) in the virus group (VR). These groups were subdivided into four subgroups: one dose analyzed 3 weeks later (VR1d3w) and two doses analyzed 1 (VR2d1w), 2 (VR2d2w) and 3 weeks (VR2d3w) after the second dose. Lung morphometry, mechanical parameters, airway responsiveness, rAAV5-GFP transduction and the expression of inflammatory cytokines were investigated. No significant differences in lung mechanics, airway responsiveness, and morphometry were observed. Re-administration of rAAV5 vector resulted in a decrease in GFP mRNA expression in the VR2d3w group. There was no evidence of inflammatory response or apoptosis in any group. rAAV5 did not induce an inflammatory process, mechanical or morphometric changes in the lungs. AAV5 may be an appropriate vector for lung gene therapy.

An increasing amount of data showing the beneficial use of zinc (Zn) in treating diarrhea continues to emerge from epidemiological and clinical trials. However, without a thorough understanding of physiological mechanisms of Zn, it does not support policy recommendation to advocate the therapeutic use of Zn. Our data demonstrate that Zn is a potential antidiarrheal agent that provides substantial benefit by stimulating sodium absorption and inhibiting chloride secretion in intestinal epithelial cells. Thus, inclusion of Zn in oral rehydration solution (ORS) has the potential to markedly augment the effectiveness of ORS in the treatment of diarrhea.

In humans, Dent's disease, an X‐linked renal tubular disorder, is characterized by low molecular weight proteinuria, aminoaciduria, glycosuria, hyperphosphaturia, hypercalciuria, nephrolithiasis, progressive renal failure and sometimes rickets or osteomalacia. The aetiology of X‐linked Dent's disease is established to be caused by mutations of the CLCN5 gene. The protein product of this gene is the voltage‐gated chloride–proton exchanger CLC‐5. Previous studies by the Johns Hopkins group (Guggino) and the Hamburg group (Jentsch) have established that the Clcn5 knockout mouse recapitulates the renal attributes of Dent's disease. In order to understand the changes in kidney function that accompany the knockout of the Clcn5 gene, we examined gene expression profiles from dissected proximal segment 1 (S1) and segment 2 (S2) tubules of mouse kidneys. Overall, 725 genes are expressed differentially in the proximal tubules of the Dent Clcn5 knockout mouse model compared with those of control wild‐type mice. A major finding is the change in the cholesterol synthesis pathway. Some interesting changes also occur in genes encoding transport proteins. One of these transport proteins, the sodium bile cotransporter gene, Slc10a2, has transcripts increased by 17‐fold in the Clcn5 knockout mouse. The Clc‐3 protein encoded by Clcn3, a chloride–proton exchanger related to Clc‐5, has a 1.9‐fold increase in transcripts. The Npt2c protein, a proximal tubule sodium phosphate cotransporter encoded by Slc34a3, has a 0.6‐fold decrease in the number of transcripts. The sodium–proton exchanger‐like protein, Nhe10/sperm, encoded by Slc9a10, has a 0.5‐fold decrease in transcript number. These genes are discussed with regard to the possible physiological outcomes of their transcript or protein changes.

Cdif is the leading cause of nosocomial diarrhea.Therapeutic interventions are failing and Cdif now accounts for more deaths in the USA than HIV.Studies on the adaptive mucosal immune response have revealed little on mechanistic pathways or new therapeutic approaches, thus in the current study we have focused on the innate immune responses to Cdif toxins.The recently described NALP family of proteins is composed of 14 members that belong to the same superfamily as NOD-2.NALPs are a component of the inflammasome complex and play critical roles in the innate immune response by recognizing conserved pathogen-associated molecular patterns.NALP/inflammasome activation involves recruitment of the adaptor protein (ASC) and triggers caspase-1-mediated cleavage of IL-1β.A role for NALPs in intestinal homeostasis and inflammation has been suggested by recent genome studies that link mutations in NALP family members and Crohn's disease.Thus, we hypothesize that activation of the inflammasome and NALP signaling plays a role in the innate immune response to Cdif toxins.Aims: Characterize NALP expression in the mouse and human colon and in intestinal epithelial and macrophage cell lines and assess the role NALP signaling in Cdif toxin-induced injury.Methods: NALP expression was assessed via qPCR.Cdif toxin-induced NALP signaling was assessed in ASC-/-and NALP-3-/-deficient macrophages by western blotting for pro-IL-1β and processed IL-1β.In Vivo studies assessed Cdif toxin-induced intestinal injury in ASC-/-mice.Results: 10 of 14 NALP transcripts were detected in human macrophages and colonic tissue, and 8 of 14 NALPs were expressed in murine macrophages and colonic tissue.In murine and human macrophages, Cdif toxin activated the inflammasome resulting in caspase-1-dependent IL-1β processing, in a doseand time-dependent fashion.Deletion of ASC, but not NALP-3, completely abolished IL-1β processing in human and murine macrophages.In Vivo, ASC-/-mice were less susceptible to Cdif toxin-induced intestinal injury and inflammation.Conclusions: This is the first study to describe NALP expression and signaling events in intestinal immune responses.Our studies revealed that inflammasome signaling events play prominent roles in the pathogenesis of Cdif toxin induced injury.Blockade of NALP/inflammasome signaling significantly reduced Cdif-induced intestinal injury and may represent a novel therapeutic approach to managing this disease.

cAMP is a major regulator of Cl − secretion. With the recognition that the exchange protein directly activated by cAMP (Epac) transduces cAMP signaling cascades independent of protein kinase A (PKA), we test the hypothesis that both PKA and Epac are involved in forskolin (FSK) stimulated Cl − secretion. FSK is an adenylate cyclase activator. T84 cells were used for short circuit current (Isc) measurement in the Ussing chamber. FSK stimulated Isc was completely inhibited by the additive effects of the PKA inhibitor, H89 (1μM) and the [Ca 2+ ]i chelator, BAPTA‐AM (25μM), suggesting the presence of both PKA dependent and independent mechanisms. Epac1 was found to be expressed in T84 cells. Both FSK and the Epac selective agonist, 8‐pCPT‐2‘‐O‐Me‐cAMP (8‐CPT, 50μM), elevated [Ca 2+ ]i. Activation of Epac1 by 8‐CPT induced Isc in intact T84 cells and in cells permeabilized with nystatin at the basolateral membrane. This effect of 8‐CPT was completely abolished by BAPTA‐AM, but not by H89 (1μM) and the CFTR specific blocker, cftr‐172, suggesting that 8‐CPT stimulated non‐CFTR channel(s) via elevation of [Ca 2+ ]i. FSK stimulated Isc was inhibited by the PLC inhibitor U73122. Both FSK and 8‐CPT increased the amount of activated (GTP‐bound) Rap proteins, which have been shown to activate PLCε to mobilize [Ca 2+ ]i. Our findings in T84 cells were further validated in mouse ileum. 8‐CPT stimulated Isc in mouse ileal sheet and this stimulation was not inhibited by H89. We conclude that PKA and Epac1 contribute to FSK stimulated Cl − secretion in intestine via PKA dependent and Ca 2+ dependent pathways, the latter mediated by Epac1‐Rap2‐PLC‐Ca 2+ signaling involving non‐CFTR channel(s).

Dent disease has multiple defects attributed to proximal tubule malfunction including low-molecular-weight proteinuria, aminoaciduria, phosphaturia, and glycosuria. To understand the changes in kidney function of the Clc5 chloride/proton exchanger gene knockout mouse model of Dent disease, we examined gene expression profiles from proximal S1 and S2 tubules of mouse kidneys. We found many changes in gene expression not known previously to be altered in this disease. Genes involved in lipid metabolism, organ development, and organismal physiological processes had the greatest number of significantly changed transcripts. In addition, genes of catalytic activity and transporter activity also had a great number of changed transcripts. Overall, 720 genes are expressed differentially in the proximal tubules of the Dent Clcn5 knockout mouse model compared with those of control wild-type mice. The fingerprint of these gene changes may help us to understand the phenotype of Dent disease.

Gene therapy using recombinant adeno-associated virus (rAAV2) vectors for cystic fibrosis has shown gene transfer and remarkable safety, yet indeterminate expression. A new construct has been characterized with a powerful exogenous promoter, the cytomegalovirus enhancer/chicken beta-actin promoter, driving a truncated CF transmembrane conductance regulator (CFTR), pseudotyped in an AAV5 viral coat. Our goal is to demonstrate that airway delivery of a pseudotyped rAAV5 vector results in gene transfer as well as expression in non-human primates. Aerosolized pseudotyped rAAV5-Delta CFTR or rAAV5-GFP (green fluorescent protein) genes were delivered to four and six lungs, respectively. The pseudotyped rAAV5 vector did result in GFP gene transfer (1.005 x 10(6) copies/mu g DNA on average) and quantifiable gene expression. Microscopy confirmed protein expression in airway epithelium. Similarly, the vector also resulted in vector-specific CFTR DNA (1.24 x 10(5) copies/mu g) and mRNA expression. Immunoprecipitation and P-32 phosphoimaging were used to demonstrate CFTR protein expression, as qualitatively enhanced beyond the barely detectable endogenous expression in untreated animals. Based on these promising studies, this CFTR minigene construct is a therapeutic candidate.

Two knockout mouse models of Dent disease are similar with regard to the characteristics of Fanconi syndrome, but differ markedly with respect to vitamin D and renal calcium handling. One model exhibits hypercalciuria, renal calcifications and renal failure; the other does not. Data from such experimental models have greatly advanced our understanding of the molecular mechanisms underlying Dent disease. This Review summarizes some of the important phenotypic characteristics shared by mouse models and people with Dent disease. Experimental data are used to predict the molecular mechanisms underlying this disease. Receptor-mediated endocytosis and the mistargeting of megalin, cubilin, the sodium/proton exchanger Nhe3 and the sodium/phosphate transporter Napi-2a will be reviewed, and the causes of mistargeting will be discussed. Kidney stones and renal failure are prominent features of Dent disease. Investigations using a mouse model with nephrocalcinosis and renal failure indicate that citrate therapy delays the onset of these processes in Dent disease. Throughout this Review, questions that might underpin new areas of investigation are proposed.

Glycosuria is one of the well-documented characteristics in ClC-5 knockout (KO) mice and patients with Dent’s disease. However, the underlying pathophysiology of its occurrence is unknown. In this study, we have compared ClC-5 KO mice with age and gender matched wild-type (WT) control mice to investigate if the underlying cause of manifested glycosuria is an impairment of glucose homeostasis and/or an alteration in expression levels of proximal tubule (PT) glucose transporters. We observed that, the blood glucose concentration (n=12, p<0.01) and the fractional excretion of glucose and insulin (n=6, p<0.05) were higher in KO mice. In contrast, the fasting blood glucose levels (n=7) were not significantly different in the two groups. Plasma glucose increased to a greater extent in KO mice (n=7, p<0.05) when challenged by an intraperitoneal injection of glucose. However, no peripheral tissue insulin resistance was observed following an intraperitoneal injection of insulin (n=9) in the KO mice. ELISA analysis demonstrated low plasma insulin concentrations after a 12 hour fasting period and also following glucose injection in KO mice. The total insulin released during a 2 hour period following glucose challenge was significantly lower in KO mice (n=6, p<0.05). By western blot, we observed a significant decrease in GLUT2 protein expression levels in isolated PT ((n=10, p<0.01)) of KO mice. This decrease in protein levels was corroborated by a significant decrease in GLUT2 mRNA levels estimated semi quantitatively by RT-PCR in isolated PT (n=10, p<0.01). No significant changes in mRNA expression levels of SGLT2, SGLT1 and GLUT1, as analyzed by RT-PCR, could be detected in the isolated PT (n=10). Also, we have shown by western blot analysis that expression of megalin is lower in the renal cortex of KO mice when compared to WT mice (n=3, p<0.05). Our results suggest that low plasma insulin concentration together with renal function changes observed in KO mice significantly contribute towards the glucose intolerance and documented glycosuria observed in this animal.

Increase in mRNA expression and transport activity of the betaine gamma-amino-n-butyric acid cotransporter (BGAT) in response to hyperosmolality has been previously shown in MDCK cells. However, the hyperosmolality-induced response of endogenous BGAT protein expression was not investigated in detail. We show two forms of endogenous BGAT immunoreactivity that are expressed in MDCK II cells. Both are sensitive to Peptide N-Glycosidase F (PNGase F), suggesting that they are N-glycosylated proteins. One band, about 75 kDa, is resistant to Endo H, while the other 55 kDa band is sensitive to it, suggesting that they are fully N-glycosylated mature form in the post-Golgi compartment and core-glycosylated immature form in the endoplasmic reticulum (ER), respectively. When treated with hyperosmolality, they are significantly increased. But the rate of BGAT processing, as assessed by the ratio of mature to immature form, is not increased, suggesting that hyperosmolality does not facilitate the export of BGAT from the ER to the secretory pathway. Surface biotinylation and confocal microscopy show that hyperosmolality significantly increases the amount of the mature form of BGAT on the basolateral membrane with a very small fraction on the apical membrane. We conclude that BGAT is an N-glycosylated protein with two glycoforms and endogenous BGAT synthesis rather than processing is involved in the adaptation to the hyperosmotic stress.

Pseudohypoaldosteronism type II (PHA II) is caused by mutations of two members of WNK ((with no lysine (k)) kinase family. WNK4 wild type (WT) has been shown to inhibit the activity and surface expression of sodium chloride cotransporter (NCC) when expressed in Xenopus oocytes. Here, we have studied NCC protein processing in mammalian cells in the presence or absence of WNK4 WT and its mutants, E562K and R1185C, by surface biotinylation, Western blot, co-immunoprecipitation (Co-IP) and immunostaining. WNK4 WT significantly reduced NCC surface expression in Cos-7 cells (58.9+/-6.8% vs 100% in control, P<0.001, n=6), whereas its mutant E562K has no significant effect on NCC surface expression (92.9+/-5.3% vs 100%, P=NS, n=6). Another mutant R1185C still partially reduces surface expression of NCC (76.2+/-11.8% vs 100%, P<0.05, n=6). The reduction of NCC surface expression by WNK4 WT (62.9+/-3.3% of control group) is not altered by WT dynamin ((61.8+/-3.7% (P=NS)) or its mutant K44A ((65.4+/-14.1% (P=NS)). A Co-IP study showed that both WNK4 WT and WNK4 E562K interact with NCC. Furthermore, a proton pump inhibitor, bafilomycin A1, partially reverses the inhibitory effect of WNK4 WT on NCC expression. Our data suggest that WNK4 WT significantly inhibits NCC surface expression, which is not owing to an increase in clathrin-mediated endocytosis of NCC, but likely results from enhanced degradation of NCC through a lysosomal pathway.