COLT: A New Weapon to Disseminate Knowledge

Too few researchers receive adequate pre- or postgraduate training to conduct a rigorous scientific study. In the digital age, new tools are emerging, and the development of distance education could improve this worrying situation. In this context, Health Science e-Training (HSeT), a nonprofit Swiss foundation, has developed new pedagogical concepts and tools under customized online training (COLT). For the ADMIRE Cost network, we have used an article-based e-learning (ABL) tool that allowed the students to learn how to read in depth and critically a scientific article and to rigorously address the problem of scientific reproducibility. The evaluation of the program by the students and the tutors has been quite positive. In conclusion COLT was well adapted to the needs of the ADMIRE Cost Action, a European network in which students from countries separated by thousands of miles can work at distance under the online supervision of tutors and then meet in a face-to-face session to maximize their learning experience and the interactions between peers and tutors

Synergistic Activation of ENaC by Three Membrane-bound Channel-activating Serine Proteases (mCAP1, mCAP2, and mCAP3) and Serum- and Glucocorticoid-regulated Kinase (Sgk1) in Xenopus Oocytes

Sodium balance is maintained by the precise regulation of the activity of the epithelial sodium channel (ENaC) in the kidney. We have recently reported an extracellular activation of ENaC-mediated sodium transport (INa) by a GPI-anchored serine protease (mouse channel–activating protein, mCAP1) that was isolated from a cortical collecting duct cell line derived from mouse kidney. In the present study, we have identified two additional membrane-bound serine proteases (mCAP2 and mCAP3) that are expressed in the same cell line. We show that each of these proteases is able to increase INa 6–10-fold in the Xenopus oocyte expression system. INa and the number (N) of channels expressed at the cell surface (measured by binding of a FLAG monoclonal I125-radioiodinated antibody) were measured in the same oocyte. Using this assay, we show that mCAP1 increases INa 10-fold (P < 0.001) but N remained unchanged (P = 0.9), indicating that mCAP1 regulates ENaC activity by increasing its average open probability of the whole cell (wcPo). The serum- and glucocorticoid-regulated kinase (Sgk1) involved in the aldosterone-dependent signaling cascade enhances INa by 2.5-fold (P < 0.001) and N by 1.6-fold (P < 0.001), indicating a dual effect on N and wcPo. Compared with Sgk1 alone, coexpression of Sgk1 with mCAP1 leads to a ninefold increase in INa (P < 0.001) and 1.3-fold in N (P < 0.02). Similar results were observed for mCAP2 and mCAP3. The synergism between CAPs and Sgk1 on INa was always more than additive, indicating a true potentiation. The synergistic effect of the two activation pathways allows a large dynamic range for ENaC-mediated sodium regulation crucial for a tight control of sodium homeostasis

Pseudohypoaldosteronisms, report on a 10-patient series

Background. Type 1 pseudohypoaldosteronism (PHA1) is a salt-wasting syndrome caused by mineralocorticoid resistance. Autosomal recessive and dominant hereditary forms are caused by Epithelial Na Channel and Mineralocorticoid Receptor mutation respectively, while secondary PHA1 is usually associated with urological problems. Methods. Ten patients were studied in four French pediatric units in order to characterize PHA1 spectrum in infants. Patients were selected by chart review. Genetic, clinical and biochemistry data were collected and analyzed. Results. Autosomal recessive PHA1 (n = 3) was diagnosed at 6 and 7 days of life in three patients presenting with severe hyperkalaemia and weight loss. After 8 months, 3 and 5 years on follow-up, neurological development and longitudinal growth was normal with high sodium supplementation. Autosomal dominant PHA1 (n = 4) was revealed at 15, 19, 22 and 30 days of life because of failure to thrive. At 8 months, 3 and 21 years of age, longitudinal growth was normal in three patients who were given salt supplementation; no significant catch-up growth was obtained in the last patient at 20 months of age. Secondary PHA1 (n = 3) was diagnosed at 11, 26 days and 5 months of life concomitantly with acute pyelonephritis in three children with either renal hypoplasia, urinary duplication or bilateral megaureter. The outcome was favourable and salt supplementation was discontinued after 3, 11 and 13 months. Conclusions. PHA1 should be suspected in case of severe hyperkalemia and weight loss in infants and need careful management. Pathogenesis of secondary PHA1 is still challenging and further studies are mandatory to highlight the link between infection, developing urinary tract and pseudohypoaldosteronis

A Novel Neutrophil Elastase Inhibitor Prevents Elastase Activation and Surface Cleavage of the Epithelial Sodium Channel Expressed in Xenopus laevis Oocytes

The amiloride-sensitive epithelial sodium channel (ENaC) constitutes a limiting step in sodium reabsorption across distal airway epithelium and controlling mucociliary clearance. ENaC is activated by serine proteases secreted in the extracellular milieu. In cystic fibrosis lungs, high concentrations of secreted neutrophil elastase (NE) are observed. hNE could activate ENaC and contribute to further decreased mucociliary clearance. The aims of this study were (i) to test the ability of an engineered human neutrophil elastase inhibitor (EPI-hNE4) to specifically inhibit the elastase activation of ENaC-mediated amiloride-sensitive currents (I(Na)) and (ii) to examine the effect of elastase on cell surface expression of ENaC and its cleavage pattern (exogenous proteolysis). Oocytes were exposed to hNE (10-100 microg/ml) and/or trypsin (10 microg/ml) for 2-5 min in the presence or absence of EPI-hNE4 (0.7 microm). hNE activated I(Na) 3.6-fold (p < 0.001) relative to non-treated hENaC-injected oocytes. EPI-hNE4 fully inhibited hNE-activated I(Na) but had no effect on trypsin- or prostasin-activated I(Na). The co-activation of I(Na) by hNE and trypsin was not additive. Biotinylation experiments revealed that cell surface gamma ENaC (but not alpha or beta ENaC) exposed to hNE for 2 min was cleaved (as a 67-kDa fragment) and correlated with increased I(Na). The elastase-induced exogenous proteolysis pattern is distinct from the endogenous proteolysis pattern induced upon preferential assembly, suggesting a causal relationship between gamma ENaC cleavage and ENaC activation, taking place at the plasma membrane

A novel TMPRSS3 missense mutation in a DFNB8/10 family prevents proteolytic activation of the protein

Pathogenic mutations in TMPRSS3, which encodes a transmembrane serine protease, cause non-syndromic deafness DFNB8/10. Missense mutations map in the low density-lipoprotein receptor A (LDLRA), scavenger-receptor cysteine-rich (SRCR), and protease domains of the protein, indicating that all domains are important for its function. TMPRSS3 undergoes proteolytic cleavage and activates the ENaC sodium channel in a Xenopus oocyte model system. To assess the importance of this gene in non-syndromic childhood or congenital deafness in Turkey, we screened for mutations affected members of 25 unrelated Turkish families. The three families with the highest LOD score for linkage to chromosome 21q22.3 were shown to harbor P404L, R216L, or Q398X mutations, suggesting that mutations in TMPRSS3 are a considerable contributor to non-syndromic deafness in the Turkish population. The mutant TMPRSS3 harboring the novel R216L missense mutation within the predicted cleavage site of the protein fails to undergo proteolytic cleavage and is unable to activate ENaC, thus providing evidence that pre-cleavage of TMPRSS3 is mandatory for normal functio

Adult nephron-specific MR-deficient mice develop a severe renal PHA-1 phenotype

Aldosterone is the main mineralocorticoid hormone controlling sodium balance, fluid homeostasis, and blood pressure by regulating sodium reabsorption in the aldosterone-sensitive distal nephron (ASDN). Germline loss-of-function mutations of the mineralocorticoid receptor (MR) in humans and in mice lead to the "renal" form of type 1 pseudohypoaldosteronism (PHA-1), a case of aldosterone resistance characterized by salt wasting, dehydration, failure to thrive, hyperkalemia, and metabolic acidosis. To investigate the importance of MR in adult epithelial cells, we generated nephron-specific MR knockout mice (MR$^{Pax8/LC1}$) using a doxycycline-inducible system. Under standard diet, MR$^{Pax8/LC1}$ mice exhibit inability to gain weight and significant weight loss compared to control mice. Interestingly, despite failure to thrive, MR$^{Pax8/LC1}$ mice survive but develop a severe PHA-1 phenotype with higher urinary Na^+ levels, decreased plasma Na(+), hyperkalemia, and higher levels of plasma aldosterone. This phenotype further worsens and becomes lethal under a sodium-deficient diet. Na^+/Cl^- co-transporter (NCC) protein expression and its phosphorylated form are downregulated in the MR$^{Pax8/LC1}$ knockouts, as well as the αENaC protein expression level, whereas the expression of glucocorticoid receptor (GR) is increased. A diet rich in Na^+ and low in K^+ does not restore plasma aldosterone to control levels but is sufficient to restore body weight, plasma, and urinary electrolytes. In conclusion, MR deletion along the nephron fully recapitulates the features of severe human PHA-1. ENaC protein expression is dependent on MR activity. Suppression of NCC under hyperkalemia predominates in a hypovolemic state

Preferential Assembly of Epithelial Sodium Channel (ENaC) Subunits in Xenopus Oocytes: ROLE OF FURIN-MEDIATED ENDOGENOUS PROTEOLYSIS

The epithelial sodium channel (ENaC) is preferentially assembled into heteromeric αβγ complexes. The α and γ (not β) subunits undergo proteolytic cleavage by endogenous furin-like activity correlating with increased ENaC function. We identified full-length subunits and their fragments at the cell surface, as well as in the intracellular pool, for all homo- and heteromeric combinations (α, β, γ, αβ, αγ, βγ, and αβγ). We assayed corresponding channel function as amiloride-sensitive sodium transport (INa). We varied furin-mediated proteolysis by mutating the P1 site in α and/or γ subunit furin consensus cleavage sites (αmut and γmut). Our findings were as follows. (i) The β subunit alone is not transported to the cell surface nor cleaved upon assembly with the α and/or γ subunits. (ii) The α subunit alone (or in combination with β and/or γ) is efficiently transported to the cell surface; a surface-expressed 65-kDa α ENaC fragment is undetected in αmutβγ, and INa is decreased by 60%. (iii) The γ subunit alone does not appear at the cell surface; γ co-expressed with α reaches the surface but is not detectably cleaved; and γ in αβγ complexes appears mainly as a 76-kDa species in the surface pool. Although basal INa of αβγmut was similar to αβγ, γmut was not detectably cleaved at the cell surface. Thus, furin-mediated cleavage is not essential for participation of α and γ in αβγ heteromers. Basal INa is reduced by preventing furin-mediated cleavage of the α, but not γ, subunits. Residual current in the absence of furin-mediated proteolysis may be due to non-furin endogenous proteases