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

PAR2 absence completely rescues inflammation and ichthyosis caused by altered CAP1/Prss8 expression in mouse skin

Altered serine protease activity is associated with skin disorders in humans and in mice. The serine protease channel-activating protease-1 (CAP1; also termed protease serine S1 family member 8 (Prss8)) is important for epidermal homeostasis and is thus indispensable for postnatal survival in mice, but its roles and effectors in skin pathology are poorly defined. In this paper, we report that transgenic expression in mouse skin of either CAP1/Prss8 (K14-CAP1/Prss8) or protease-activated receptor-2 (PAR2; Grhl3PAR2/+), one candidate downstream target, causes epidermal hyperplasia, ichthyosis and itching. K14-CAP1/Prss8 ectopic expression impairs epidermal barrier function and causes skin inflammation characterized by an increase in thymic stromal lymphopoietin levels and immune cell infiltrations. Strikingly, both gross and functional K14-CAP1/Prss8-induced phenotypes are completely negated when superimposed on a PAR2-null background, establishing PAR2 as a pivotal mediator of pathogenesis. Our data provide genetic evidence for PAR2 as a downstream effector of CAP1/Prss8 in a signalling cascade that may provide novel therapeutic targets for ichthyoses, pruritus and inflammatory skin diseases

Effetti della acetil-L-carnitina sulle cellule sensoriali T della sanguisuga "Hirudo medicinalis"

L’acetil-L-carnitina (ALC) è una molecola presente nel sistema nervoso in grado di influenzare molti aspetti del metabolismo neuronale e di migliorare la funzionalità dei circuiti neurali. Fino ad oggi, però, non sono stati descritti, in dettaglio, effetti di ALC sulle proprietà elettriche di singoli neuroni. In questa tesi sperimentale, è stata indagata l’azione di ALC sui neuroni sensoriali T della sanguisuga "Hirudo medicinalis", invertebrato ampiamente utilizzato per indagini elettrofisiologiche, neurochimiche e comportamentali sui vari aspetti dell’attività neurale. Ogni ganglio segmentale di "H. medicinalis" contiene circa 400 neuroni, inclusi i meccanocettori tattili T, che possono essere agevolmente identificati. La stimolazione dei loro campi recettoriali induce, nei neuroni T, treni di potenziali d’azione accompagnati da un’ampia iperpolarizzazione postuma (AHP) dovuta principalmente all’attività della pompa Na+/K+ ATPasi e parzialmente ad una conduttanza K+/Ca2+-dipendente (gKCa). AHP può essere indotta, inoltre, iniettando intracellularmente treni di impulsi depolarizzanti della durata di 3 sec (2.5 Hz di frequenza) ad una intensità di corrente variabile tra 0.4 - 0.8 nA. Nelle cellule T, i cambiamenti dell’ampiezza di AHP possono essere direttamente correlati a modificazioni dell’attività della pompa Na+/K+ ATPasi. Precedentemente, è stato osservato che, a livello comportamentale, ALC influenza due tipi di apprendimento non associativo: blocca la sensitizzazione e diminuisce la disabitudine nell’induzione al nuoto. A livello cellulare, registrando neuroni T di gangli isolati da animali iniettati 6 giorni prima con ALC 2 mM, si è osservato che la sostanza produce un significativo aumento dell’ampiezza di AHP, una iperpolarizzazione del voltaggio di membrana (Vm) e un aumento della resistenza di ingresso (Rm). Allo scopo di investigare il meccanismo molecolare con cui ALC agisce, sono state condotte registrazioni elettrofisiologiche in current-clamp convenzionale da neuroni T di gangli estratti da animali naïves. La perfusione per 10 minuti con ALC a concentrazioni comprese tra 5 e 50 microM ha evidenziato effetti dose-dipendenti su AHP, Vm, mentre non ha prodotto modificazioni di Rm; alla concentrazione di 50 microM, ALC determina gli stessi effetti osservati nelle cellule T dei gangli estratti dagli animali precedentemente iniettati con la sostanza. Concentrazioni di ALC maggiori (100 microM), invece, diminuiscono l’ampiezza di AHP senza influenzare Vm o Rm. Gli effetti di ALC 50 microM permangono anche in soluzione di alto magnesio (20 mM), utilizzato per bloccare la trasmissione sinaptica chimica. Quando applicato da solo, il magnesio non modifica AHP. Ciò suggerisce un’azione diretta di ALC sui neuroni T e, più precisamente, sui meccanismi responsabili di AHP. Per verificare questa ipotesi, sono state condotte due serie di esperimenti. Nella prima serie, è stata bloccata gKCa con apamina. ALC 50 microM si è mostrata in grado di determinare un incremento di AHP anche quando gKCa è bloccata. Questo risultato suggerisce un effetto di ALC sull’altra componente responsabile della genesi di AHP, la Na+/K+ ATPasi. Nella seconda serie di esperimenti, per valutare se effettivamente ALC moduli l’attività della Na+/K+ ATPasi, le cellule T sono state iniettate con sodio (usando microelettrodi riempiti con sodio acetato 2 M) che aumenta la funzionalità della pompa determinando una iperpolarizzazione di Vm. L’iniezione di sodio in neuroni T trattati con ALC 50 microM determina una maggiore iperpolarizzazione di Vm. ALC è quindi capace di modulare positivamente l’attività della Na+/K+ ATPasi. Resta da chiarire se il target molecolare di ALC sia proprio la Na+/K+ ATPasi o se questa sostanza esplichi la sua azione in modo indiretto, attraverso la modulazione di altri elementi proteici cellulari

ENaC-mediated alveolar fluid clearance and lung fluid balance depend on the channel-activating protease 1.

Sodium transport via epithelial sodium channels (ENaC) expressed in alveolar epithelial cells (AEC) provides the driving force for removal of fluid from the alveolar space. The membrane-bound channel-activating protease 1 (CAP1/Prss8) activates ENaC in vitro in various expression systems. To study the role of CAP1/Prss8 in alveolar sodium transport and lung fluid balance in vivo, we generated mice lacking CAP1/Prss8 in the alveolar epithelium using conditional Cre-loxP-mediated recombination. Deficiency of CAP1/Prss8 in AEC induced in vitro a 40% decrease in ENaC-mediated sodium currents. Sodium-driven alveolar fluid clearance (AFC) was reduced in CAP1/Prss8-deficient mice, due to a 48% decrease in amiloride-sensitive clearance, and was less sensitive to beta(2)-agonist treatment. Intra-alveolar treatment with neutrophil elastase, a soluble serine protease activating ENaC at the cell surface, fully restored basal AFC and the stimulation by beta(2)-agonists. Finally, acute volume-overload increased alveolar lining fluid volume in CAP1/Prss8-deficient mice. This study reveals that CAP1 plays a crucial role in the regulation of ENaC-mediated alveolar sodium and water transport and in mouse lung fluid balance

Lack of Renal Tubular Glucocorticoid Receptor Decreases the Thiazide-Sensitive Na+/Cl– Cotransporter NCC and Transiently Affects Sodium Handling

International audienceChronic glucocorticoid infusion impairs NCC activity and induces a non-dipping profile in mice, suggesting that glucocorticoids are essential for daily blood pressure variations. In this paper, we studied mice lacking the renal tubular glucocorticoid receptor (GR) in adulthood (GR knockouts, Nr3c1Pax8/LC1). Upon standard salt diet, Nr3c1Pax8/LC1 mice grow normally, but show reduced NCC activity despite normal plasma aldosterone levels. Following diet switch to low sodium, Nr3c1Pax8/LC1 mice exhibit a transient but significant reduction in the activity of NCC and expression of NHE3 and NKCC2 accompanied by significant increased Spak activity. This is followed by transiently increased urinary sodium excretion and higher plasma aldosterone concentrations. Plasma corticosterone levels and 11βHSD2 mRNA expression and activity in the whole kidney remain unchanged. High salt diet does not affect whole body Na+ and/or K+ balance and NCC activity is not reduced, but leads to a significant increase in diastolic blood pressure dipping in Nr3c1Pax8/LC1 mice. When high sodium treatment is followed by 48 h of darkness, NCC abundance is reduced in knockout mice although activity is not different. Our data show that upon Na+ restriction renal tubular GR-deficiency transiently affects Na+ handling and transport pathways. Overall, upon standard, low Na+ and high Na+ diet exposure Na+ and K+ balance is maintained as evidenced by normal plasma and urinary Na+ and K+ and aldosterone concentrations

CAP1/Prss8 knockout embryos are born and exhibit an abnormal epidermis.

<p>Genotype distribution of pups at birth and at weaning (<i>CAP1/Prss8</i>^Δ<i>/+</i><i>;Sox2::Cre^Tg/+</i> x <i>CAP1/Prss8^lox/lox</i>). Two litters were taken right after birth (n = 21 pups, <b>A</b>) or following weaning (3 week-old; n = 26 pups, <b>B</b>) and genotyped. (<b>C</b>) H&E staining of sagittal backskin sections of one-day-old pups from controls (<i>CAP1/Prss8^lox/+</i>; Lox/+ cre-), heterozygous (<i>CAP1/Prss8^lox/</i>^Δ; Lox/Δ cre-) and embryo-specific knockouts (<i>CAP1/Prss8^lox/</i>^Δ<i>;Sox2::Cre^Tg/+</i>; Lox/Δ cre+). The latter displayed a compact stratum corneum (hyperkeratosis) indicative of a skin barrier defect. Four additional litters were genotyped at weaning; magnification x200.</p

CAP1/Prss8 knockout placentas present a late differentiation defect.

<p>Representative pictures of placenta. (<b>A</b>) Placental discs at E12.5 and E14.5. H&E stainings of sagittal sections of placentas at E12.5 and E14.5 from wildtype (WT), heterozygous (Het) and knockout (KO) embryos at (<b>B</b>) low (x50) and at (<b>C</b>) high magnification (x400). From day E12.5 onwards, maturation from cytotrophoblasts to syncytiotrophoblasts is observed in wildtype and heterozygous mutant but not in knockout. The dashed line indicates the limit between the labyrinth and the decidua in <b>B</b>, and surrounds the cytotrophoblasts in <b>C</b>.</p

CAP1/Prss8 knockout embryos present normal placentas.

<p>Epiblast-specific CAP1/Prss8 knockout (Lox/Δ cre+) pups were obtained by mating <i>CAP1/Prss8</i>^Δ<i>/+</i><i>;Sox2::Cre^Tg/+</i> to <i>CAP1/Prss8^lox/lox</i> mice. (<b>A</b>) Genotype distribution of embryos recovered at E14.5 and relative quantification of CAP1/Prss8 mRNA expression levels in placentas and embryos (<b>B</b>). <i>CAP1/Prss8^lox/</i>^Δ<i>;Sox2::Cre^Tg/+</i>, embryo-specific knockout (Lox/Δ Cre+), white bar; <i>CAP1/Prss8^lox/</i>^Δ (Lox/Δ Cre-) and <i>CAP1/Prss8^lox/+;Sox2::Cre^Tg/+</i>, heterozygous mutant (Lox/+ Cre+), grey bar; <i>CAP1/Prss8^lox/+</i> wildtype (Lox/+ Cre-); black bar. Note that the CAP1/Prss8 mRNA expression in the knockout (Lox/Δ Cre+) embryos is abolished while the corresponding control (Lox/+ Cre+) placentas show heterozygous expression levels. (<b>C</b>) H&E staining of sagittal placenta sections at E14.5 from controls (<i>CAP1/Prss8^lox/+</i>; Lox/+ cre-), heterozygotes (<i>CAP1/Prss8^lox/</i>^Δ; Lox/Δ) and embryo-specific knockouts (<i>CAP1/Prss8^lox/</i>^Δ<i>;Sox2::Cre^Tg/+</i>; Lox/Δ cre+).</p