Amiloride-sensitive epithelial sodium channels (ENaC) play an important role in lung sodium transport. Sodium transport is closely regulated to maintain an appropriate fluid layer on the alveolar surface. Both alveolar type I and II cells have several different sodium-permeable channels in their apical membranes that play a role in normal lung physiology and pathophysiology. In many epithelial tissues, ENaC is formed from three subunit proteins: alpha, beta, and gamma ENaC. Part of the diversity of sodium-permeable channels in lung arises from assembling different combinations of these subunits to form channels with different biophysical properties and different mechanisms for regulation. Thus, lung epithelium has enormous flexibility to alter the magnitude of salt and water transport. In lung, ENaC is regulated by many transmitter and hormonal agents. Regulation depends upon the type of sodium channel but involves controlling the number of apical channels and/or the activity of individual channels.

Rationale: Alveolar liquid clearance is regulated by Na 1 uptake through the apically expressed epithelial sodium channel (ENaC) and basolaterally localized Na 1 -K 1 -ATPase in type II alveolar epithelial cells. Dysfunction of these Na 1 transporters during pulmonary inflammation can contribute to pulmonary edema.Objectives: In this study, we sought to determine the precise mechanism by which the TIP peptide, mimicking the lectin-like domain of tumor necrosis factor (TNF), stimulates Na 1 uptake in a homologous cell system in the presence or absence of the bacterial toxin pneumolysin (PLY).Methods: We used a combined biochemical, electrophysiological, and molecular biological in vitro approach and assessed the physiological relevance of the lectin-like domain of TNF in alveolar liquid clearance in vivo by generating triple-mutant TNF knock-in mice that express a mutant TNF with deficient Na 1 uptake stimulatory activity.Measurements and Main Results: TIP peptide directly activates ENaC, but not the Na 1 -K 1 -ATPase, upon binding to the carboxyterminal domain of the a subunit of the channel. In the presence of PLY, a mediator of pneumococcal-induced pulmonary edema, this binding stabilizes the ENaC-PIP2-MARCKS complex, which is necessary for the open probability conformation of the channel and preserves ENaC-a protein expression, by means of blunting the protein kinase C-a pathway. Triple-mutant TNF knock-in mice are more prone than wild-type mice to develop edema with low-dose intratracheal PLY, correlating with reduced pulmonary ENaC-a subunit expression.Conclusions: These results demonstrate a novel TNF-mediated mechanism of direct ENaC activation and indicate a physiological role for the lectin-like domain of TNF in the resolution of alveolar edema during inflammation.

Evidence suggests that glycogen synthase kinase 3b (GSK3b) contributes to AKI; however, its role in post-AKI kidney repair remains uncertain. Here, delayed treatment with a single dose of lithium, a selective inhibitor of GSK3b and a US Food and Drug Administration-approved mood stabilizer, accelerated recovery of renal function, promoted repopulation of renal tubular epithelia, and improved kidney repair in murine models of cisplatin-and ischemia/reperfusion-induced AKI. These effects associated with reduced GSK3b activity and elevated expression of proproliferative molecules, including cyclin D1, c-Myc, and hypoxia-inducible factor 1a (HIF-1a), in renal tubular epithelia. In cultured renal tubular cells, cisplatin exposure led to transient repression of GSK3b activity followed by a prolonged upregulation of activity. Rescue treatment with lithium inhibited GSK3b activity, enhanced nuclear expression of cyclin D1, c-Myc, and HIF-1a, and boosted cellular proliferation. Similarly, ectopic expression of a kinase-dead mutant of GSK3b enhanced the expression of cyclin D1, c-Myc, and HIF-1a and amplified cellular proliferation after cisplatin injury, whereas forced expression of a constitutively active mutant of GSK3b abrogated the effects of lithium. Mechanistically, GSK3b colocalized and physically interacted with cyclin D1, c-Myc, and HIF-1a in tubular cells. In silico analysis revealed that cyclin D1, c-Myc, and HIF-1a harbor putative GSK3b consensus phosphorylation motifs, implying GSK3b-directed phosphorylation and subsequent degradation of these molecules. Notably, cotreatment with lithium enhanced the proapoptotic effects of cisplatin in cultured colon cancer cells. Collectively, our findings suggest that pharmacologic targeting of GSK3b by lithium may be a novel therapeutic strategy to improve renal salvage after AKI.

The bicyclic fatty acid lubiprostone (formerly known as SPI-0211) activates two types of anion channels in A6 cells. Both channel types are rarely, if ever, observed in untreated cells. The first channel type was activated at low concentrations of lubiprostone (<100 nM) in >80% of cell-attached patches and had a unit conductance of approximately 3-4 pS. The second channel type required higher concentrations (>100 nM) of lubiprostone to activate, was observed in approximately 30% of patches, and had a unit conductance of 8-9 pS. The properties of the first type of channel were consistent with ClC-2 and the second with CFTR. ClC-2's unit current strongly inwardly rectified that could be best fit by models of the channel with multiple energy barrier and multiple anion binding sites in the conductance pore. The open probability and mean open time of ClC-2 was voltage dependent, decreasing dramatically as the patches were depolarized. The order of anion selectivity for ClC-2 was Cl > Br > NO(3) > I > SCN, where SCN is thiocyanate. ClC-2 was a "double-barreled" channel favoring even numbers of levels over odd numbers as if the channel protein had two conductance pathways that opened independently of one another. The channel could be, at least, partially blocked by glibenclamide. The properties of the channel in A6 cells were indistinguishable from ClC-2 channels stably transfected in HEK293 cells. CFTR in the patches had a selectivity of Cl > Br >> NO(3) congruent with SCN congruent with I. It outwardly rectified as expected for a single-site anion channel. Because of its properties, ClC-2 is uniquely suitable to promote anion secretion with little anion reabsorption. CFTR, on the other hand, could promote either reabsorption or secretion depending on the anion driving forces.

Phosphatidylinositol phosphates (PIPs) are known to regulate epithelial sodium channels (ENaC). Lipid binding assays and coimmunoprecipitation showed that the amino-terminal domain of the β- and γ-subunits of Xenopus ENaC can directly bind to phosphatidylinositol 4,5-bisphosphate (PIP(2)), phosphatidylinositol 3,4,5-trisphosphate (PIP(3)), and phosphatidic acid (PA). Similar assays demonstrated various PIPs can bind strongly to a native myristoylated alanine-rich C-kinase substrate (MARCKS), but weakly or not at all to a mutant form of MARCKS. Confocal microscopy demonstrated colocalization between MARCKS and PIP(2). Confocal microscopy also showed that MARCKS redistributes from the apical membrane to the cytoplasm after PMA-induced MARCKS phosphorylation or ionomycin-induced intracellular calcium increases. Fluorescence resonance energy transfer studies revealed ENaC and MARCKS in close proximity in 2F3 cells when PKC activity and intracellular calcium concentrations are low. Transepithelial current measurements from Xenopus 2F3 cells treated with PMA and single-channel patch-clamp studies of Xenopus 2F3 cells treated with a PKC inhibitor altered Xenopus ENaC activity, which suggest an essential role for MARCKS in the regulation of Xenopus ENaC activity.

Objective Impaired adaptive response to oxidative injuries is a fundamental mechanism central to the pathogenesis of chronic hepatitis C (CHC). Glycogen synthase kinase (GSK) 3β is an indispensable regulator of the oxidative stress response. However, the exact role of GSK3β in CHC is uncertain and was examined. Design GSK3β and Nrf2 signaling pathways were examined in JFH1 hepatitis C virus (HCV) infected Huh 7.5.1 hepatocytes and also in liver biopsy specimens from CHC patients. Results HCV infection elicited prominent Nrf2 antioxidant response in hepatocytes, marked by elevated expression of the Nrf2 dependent molecule heme oxygenase-1 and subsequent protection from apoptotic cell death. Inhibitory phosphorylation of GSK3β seems to be essential and sufficient for HCV induced Nrf2 response. Mechanistically, GSK3β physically associated and interacted with Nrf2 in hepatocytes. In silico analysis revealed that Nrf2 encompasses multiple GSK3β phosphorylation consensus motifs, denoting Nrf2 as a cognate substrate of GSK3β. In the presence of TGFβ1, the HCV induced GSK3β phosphorylation was blunted via a protein phosphatase 1-dependent mechanism and the cytoprotective Nrf2 response drastically impaired. Lithium, a selective inhibitor of GSK3β, counteracted the effects of TGFβ1. In liver biopsy specimens from CHC patients, the expression of phosphorylated GSK3β positively correlated with Nrf2 expression and was inversely associated with the degree of liver injury. Moreover, CHC patients who received long-term lithium carbonate therapy primarily for concomitant psychiatric disorders exhibited much less liver injury, associated with enhanced hepatic expression of Nrf2. Conclusions Inhibition of GSK3β exerts hepatoprotection in CHC possibly through its direct regulation of Nrf2 antioxidant response.

NFκB is regulated by a myriad of signaling cascades including glycogen synthase kinase (GSK) 3β and plays a Janus role in podocyte injury. In vitro, lipopolysaccharide or adriamycin elicited podocyte injury and cytoskeletal disruption, associated with NFκB activation and induced expression of NFκB target molecules, including pro-survival Bcl-xL and podocytopathic mediators like MCP-1, cathepsin L and B7-1. Broad range inhibition of NFκB diminished the expression of all NFκB target genes, restored cytoskeleton integrity, but potentiated apoptosis. In contrast, blockade of GSK3β by lithium or TDZD-8, mitigated the expression of podocytopathic mediators, ameliorated podocyte injury, but barely affected Bcl-xL expression or sensitized apoptosis. Mechanistically, GSK3β was sufficient and essential for RelA/p65 phosphorylation specifically at serine 467, which specifies the expression of selective NFκB target molecules, including podocytopathic mediators, but not Bcl-xL. In vivo, lithium or TDZD-8 therapy improved podocyte injury and proteinuria in mice treated with lipopolysaccharide or adriamycin, concomitant with suppression of podocytopathic mediators but retained Bcl-xL in glomerulus. Broad range inhibition of NFκB conferred similar but much weakened antiproteinuric and podoprotective effects accompanied with a blunted glomerular expression of Bcl-xL and marked podocyte apoptosis. Thus, the GSK3β dictated fine-tuning of NFκB may serve as a novel therapeutic target for podocytopathy.

Active Na+ reabsorption by alveolar epithelial cells generates the driving force used to clear fluids from the air space. Using single-channel methods, we examined epithelial Na+ channel (ENaC) activity of alveolar type I (AT1) cells from live 250- to 300-microm sections of lung tissue, circumventing concerns that protracted cell isolation procedures might compromise the innate transport properties of native lung cells. We used fluorescein-labeled Erythrina crystagalli lectin to positively identify AT1 cells for single-channel patch-clamp analysis. We demonstrated, for the first time, single-channel recordings of highly selective and nonselective amiloride-sensitive ENaC channels (HSC and NSC, respectively) from AT1 cells in situ, with mean conductances of 8.2+/-2.5 and 22+/-3.2 pS, respectively. Additionally, 25 nM amiloride in the patch electrode blocked Na+ channel activity in AT1 cells. Immunohistochemical studies demonstrated the presence of dopamine D1 and D2 receptors on the surface of AT1 cells, and single-channel recordings showed that 10 microM dopamine increased Na+ channel activity [product of the number of channels and single-channel open probability (NPo)] from 0.31+/-0.19 to 0.60+/-0.21 (P<0.001). The D1 receptor antagonist SCH-23390 (10 microM) blocked the stimulatory effect of dopamine on AT1 cells, but the D2 receptor antagonist sulpiride did not.