Anuwat Dinudom

Pathogenesis of chytridiomycosis, a cause of catastrophic amphibian declines.

Croaking Frogs The global amphibian decline has been attributed, among other causes, to an amphibian skin disease chytridiomycosis caused by the fungus Batrachochytrium dendrobatidis. However, how this pathogen causes mortality has been unclear. Voyles et al. (p. 582) show that this superficial skin infection may lead to cardiac failure owing to changes caused by lowered ion transport through the skin and consequent electrolyte reduction in the blood. A fungal disease that is associated with frog mortality causes changes in electrolyte transport across the skin. The pathogen Batrachochytrium dendrobatidis (Bd), which causes the skin disease chytridiomycosis, is one of the few highly virulent fungi in vertebrates and has been implicated in worldwide amphibian declines. However, the mechanism by which Bd causes death has not been determined. We show that Bd infection is associated with pathophysiological changes that lead to mortality in green tree frogs (Litoria caerulea). In diseased individuals, electrolyte transport across the epidermis was inhibited by >50%, plasma sodium and potassium concentrations were respectively reduced by ~20% and ~50%, and asystolic cardiac arrest resulted in death. Because the skin is critical in maintaining amphibian homeostasis, disruption to cutaneous function may be the mechanism by which Bd produces morbidity and mortality across a wide range of phylogenetically distant amphibian taxa.

Increased Gut Permeability and Microbiota Change Associate with Mesenteric Fat Inflammation and Metabolic Dysfunction in Diet-Induced Obese Mice

We investigated the relationship between gut health, visceral fat dysfunction and metabolic disorders in diet-induced obesity. C57BL/6J mice were fed control or high saturated fat diet (HFD). Circulating glucose, insulin and inflammatory markers were measured. Proximal colon barrier function was assessed by measuring transepithelial resistance and mRNA expression of tight-junction proteins. Gut microbiota profile was determined by 16S rDNA pyrosequencing. Tumor necrosis factor (TNF)-α and interleukin (IL)-6 mRNA levels were measured in proximal colon, adipose tissue and liver using RT-qPCR. Adipose macrophage infiltration (F4/80+) was assessed using immunohistochemical staining. HFD mice had a higher insulin/glucose ratio (P = 0.020) and serum levels of serum amyloid A3 (131%; P = 0.008) but reduced circulating adiponectin (64%; P = 0.011). In proximal colon of HFD mice compared to mice fed the control diet, transepithelial resistance and mRNA expression of zona occludens 1 were reduced by 38% (P<0.001) and 40% (P = 0.025) respectively and TNF-α mRNA level was 6.6-fold higher (P = 0.037). HFD reduced Lactobacillus (75%; P<0.001) but increased Oscillibacter (279%; P = 0.004) in fecal microbiota. Correlations were found between abundances of Lactobacillus (r = 0.52; P = 0.013) and Oscillibacter (r = −0.55; P = 0.007) with transepithelial resistance of the proximal colon. HFD increased macrophage infiltration (58%; P = 0.020), TNF-α (2.5-fold, P<0.001) and IL-6 mRNA levels (2.5-fold; P = 0.008) in mesenteric fat. Increased macrophage infiltration in epididymal fat was also observed with HFD feeding (71%; P = 0.006) but neither TNF-α nor IL-6 was altered. Perirenal and subcutaneous adipose tissue showed no signs of inflammation in HFD mice. The current results implicate gut dysfunction, and attendant inflammation of contiguous adipose, as salient features of the metabolic dysregulation of diet-induced obesity.

The Nedd4-like Protein KIAA0439 Is a Potential Regulator of the Epithelial Sodium Channel

The amiloride-sensitive epithelial sodium channel (ENaC) plays a critical role in fluid and electrolyte homeostasis and consists of α, β, and γ subunits. The carboxyl terminus of each ENaC subunit contains a PPxY, motif which is believed to be important for interaction with the WW domains of the ubiquitin-protein ligase, Nedd4. Disruption of this interaction, as in Liddles syndrome, where mutations delete or alter the PPxY motif of either the β or γ subunits, has been proposed to result in increased ENaC activity. Here we present evidence that KIAA0439 protein, a close relative of Nedd4, is also a potential regulator of ENaC. We demonstrate that KIAA0439 WW domains bind all three ENaC subunits. We show that a recombinant KIAA0439 WW domain protein acts as a dominant negative mutant that can interfere with the Na+-dependent feedback inhibition of ENaC in whole-cell patch clamp experiments. We propose that KIAA0439 and Nedd4 proteins either play a redundant role in ENaC regulation or function in a tissue- and/or signal-specific manner to down-regulate ENaC.

All Three WW Domains of Murine Nedd4 Are Involved in the Regulation of Epithelial Sodium Channels by Intracellular Na

The amiloride-sensitive epithelial sodium channel (ENaC) plays a critical role in fluid and electrolyte homeostasis and consists of α, β, and γ subunits. The carboxyl terminus of each ENaC subunit contains a PPxY motif which is necessary for interaction with the WW domains of the ubiquitin-protein ligase, Nedd4. Disruption of this interaction, as in Liddle’s syndrome where mutations delete or alter the PY motif of either the β or γ subunits, results in increased ENaC activity. We have recently shown using the whole-cell patch clamp technique that Nedd4 mediates the ubiquitin-dependent down-regulation of Na+ channel activity in response to increased intracellular Na+. In this paper, we demonstrate that WW domains 2 and 3 bind α-, β-, and γ-ENaC with varying degrees of affinity, whereas WW domain 1 does not bind to any of the subunits. We further show using whole-cell patch clamp techniques that Nedd4-mediated down-regulation of ENaC in mouse mandibular duct cells involves binding of the WW domains of Nedd4 to three distinct sites. We propose that Nedd4-mediated down-regulation of Na+ channels involves the binding of WW domains 2 and 3 to the Na+channel and of WW domain 1 to an unknown associated protein.

Akt mediates the effect of insulin on epithelial sodium channels by inhibiting Nedd4-2

The epithelial sodium channel (ENaC) plays an important role in transepithelial Na+ absorption; hence its function is essential for maintaining Na+ and fluid homeostasis and regulating blood pressure. Insulin is one of the hormones that regulates activity of ENaC. In this study, we investigated the contribution of two related protein kinases, Akt (also known as protein kinase B) and the serum- and glucocorticoid-dependent kinase (Sgk), on insulin-induced ENaC activity in Fisher rat thyroid cells expressing ENaC. Overexpression of Akt1 or Sgk1 significantly increased ENaC activity, whereas expression of a dominant-negative construct of Akt1, Akt1K179M, decreased basal activity of ENaC. Inhibition of the endogenous expression of Akt1 and Sgk1 by short interfering RNA not only inhibited ENaC but also disrupted the stimulatory effect on ENaC of insulin and of the downstream effectors of insulin, phosphatidylinositol 3-kinase and PDK1. Conversely, overexpression of Akt1 or Sgk1 increased expression of ENaC at the cell membrane and overcame the inhibitory effect of Nedd4-2 on ENaC. Furthermore, mutation of consensus phosphorylation sites on Nedd4-2 for Akt1 and Sgk1, Ser342 and Ser428, completely abolished the inhibitory effect of Sgk1 and Akt1 on Nedd4-2 action. Together these data suggest that both Akt and Sgk are components of an insulin signaling pathway that increases Na+ absorption by up-regulating membrane expression of ENaC via a regulatory system that involves inhibition of Nedd4-2.

Na+ and Cl− conductances are controlled by cytosolic Cl− concentration in the intralobular duct cells of mouse mandibular glands

Our previously published whole-cell patch-clamp studies on the cells of the intralobular (granular) ducts of the mandibular glands of male mice revealed the presence of an amiloride-sensitive Na+ conductance in the plasma membrane. In this study we demonstrate the presence also of a Cl− conductance and we show that the sizes of both conductances vary with the Cl− concentration of the fluid bathing the cytosolic surface of the plasma membrane. As the cytosolic Cl− concentration rises from 5 to 150 mmol/liter, the size of the inward Na+ current declines, the decline being half-maximal when the Cl− concentration is approximately 50 mmol/liter. In contrast, as cytosolic Cl− concentration increases, the inward Cl− current remains at a constant low level until the Cl− concentration exceeds 80 mmol/liter, when it begins to increase. Studies in which Cl− in the pipette solution was replaced by other anions indicate that the Na+ current is suppressed by intracellular Br-, Cl− and NO3-but not by intracellular I-, glutamate or gluconate. Our studies also show that the Cl− conductance allows passage of Cl− and Br- equally well, I-less well, and NO3-, glutamate and gluconate poorly, if at all. The findings with NO3-are of particular interest because they show that suppression of the Na+ current by a high intracellular concentration of a particular anion does not depend on actual passage of that anion through the Cl− conductance. In mouse granular duct cells there is, thus, a reciprocal regulation of Na+ and Cl− conductances by the cytosolic Cl− concentration. Since the cytosolic Cl− concentration is closely correlated with cell volume in many epithelia, this reciprocal regulation of Na+ and Cl− conductances may provide a mechanism by which ductal Na+ and Cl transport rates are adjusted so as to maintain a stable cell volume.

The role of individual Nedd4–2 (KIAA0439) WW domains in binding and regulating epithelial sodium channels

The amiloride‐sensitive epithelial sodium channel (ENaC) is essential for fluid and electrolyte homeostasis. ENaC consists of α, β, and γ subunits, each of which contains a PPxY motif that interacts with the WW domains of the ubiquitin‐protein ligases Nedd4 and Nedd4–2. Disruption of this interaction, as in Liddles syndrome in which mutations delete or alter the PPxY motif of either the β or the γ subunits, results in increased ENaC activity. We report here that Nedd4–2 has two major isoforms that show tissue‐specific expression; however, both isoforms can inhibit ENaC in Xenopus oocytes. Because there are four WW domains in Nedd4–2, we analyzed binding kinetics and affinity between individual WW domains and ENaC subunits. Using whole cell patch‐clamp techniques, we studied the role of individual WW domains in the regulation of ENaC in mammalian cells. We report here that unlike Nedd4, only two of the Nedd4–2 WW domains, WW3 and WW4, are required for both the binding to ENaC subunits and the regulation of Na+ feedback control of ENaC. Although both WW3 and WW4 individually can interact with all three ENaC subunits in vitro, both domains together are essential for in vivo function of Nedd4–2 in ENaC regulation. These data suggest that Nedd4–2 WW3 and WW4 interact with distinct, noninterchangeable sites in ENaC and that to prevent Na+ feedback control of ENaC it is necessary to occlude both sites.

Respiratory distress and perinatal lethality in Nedd4-2-deficient mice

The epithelial sodium channel (ENaC) is essential for sodium homoeostasis in many epithelia. ENaC activity is required for lung fluid clearance in newborn animals and for maintenance of blood volume and blood pressure in adults. In vitro studies show that the ubiquitin ligase Nedd4-2 ubiquitinates ENaC to regulate its cell surface expression. Here we show that knockout of Nedd4-2 in mice leads to increased ENaC expression and activity in embryonic lung. This increased ENaC activity is the likely reason for premature fetal lung fluid clearance in Nedd4-2−/− animals, resulting in a failure to inflate lungs and perinatal lethality. A small percentage of Nedd4-2−/− animals survive up to 22 days, and these animals also show increased ENaC expression and develop lethal sterile inflammation of the lung. Thus, we provide critical in vivo evidence that Nedd4-2 is essential for correct regulation of ENaC expression, fetal and postnatal lung function and animal survival.

Control of the amiloride‐sensitive Na+ current in mouse salivary ducts by intracellular anions is mediated by a G protein.

1. We have previously reported that the Na+ conductance in mouse intralobular salivary duct cells is controlled by cytosolic anions, being inhibited by high cytosolic concentrations of Cl‐ and NO3‐ but not of glutamate. In the present paper, we use whole‐cell patch‐clamp methods to investigate whether this anion effect is mediated by a G protein. 2. Inclusion of 100 mumol l‐1 GTP‐gamma‐S, a non‐hydrolysable GTP analogue, in the glutamate‐containing pipette solution, i.e. when the Na+ conductance is active, reduced the size of the Na+ conductance whereas inclusion of 100 mumol l‐1 GDP‐beta‐S, a non‐hydrolysable GDP analogue, had no effect. 3. Inclusion of 100 mumol l‐1 GDP‐beta‐S in the NO3(‐)‐containing pipette solution, i.e. when the Na+ conductance is inhibited, reactivated the conductance. Inclusion of 500 ng ml‐1 activated pertussis toxin in the NO3(‐)‐containing pipette solution had a similar effect on the Na+ conductance. 4. We conclude that the inhibitory effect of intracellular anions such as NO3‐ and Cl‐ on the amiloride‐sensitive Na+ conductance in mouse mandibular intralobular duct cells is mediated by a G protein sensitive to pertussis toxin.

The Activity of the Epithelial Sodium Channels Is Regulated by Caveolin-1 via a Nedd4-2-dependent Mechanism

It has recently been shown that the epithelial Na+ channel (ENaC) is compartmentalized in caveolin-rich lipid rafts and that pharmacological depletion of membrane cholesterol, which disrupts lipid raft formation, decreases the activity of ENaC. Here we show, for the first time, that a signature protein of caveolae, caveolin-1 (Cav-1), down-regulates the activity and membrane surface expression of ENaC. Physical interaction between ENaC and Cav-1 was also confirmed in a coimmunoprecipitation assay. We found that the effect of Cav-1 on ENaC requires the activity of Nedd4-2, a ubiquitin protein ligase of the Nedd4 family, which is known to induce ubiquitination and internalization of ENaC. The effect of Cav-1 on ENaC requires the proline-rich motifs at the C termini of the β- and γ-subunits of ENaC, the binding motifs that mediate interaction with Nedd4-2. Taken together, our data suggest that Cav-1 inhibits the activity of ENaC by decreasing expression of ENaC at the cell membrane via a mechanism that involves the promotion of Nedd4-2-dependent internalization of the channel.