Measurements of spontaneous CFTR-mediated ion transport without acute channel activation in airway epithelial cultures after modulator exposure

Quantitation of CFTR function in vitro is commonly performed by acutely stimulating then inhibiting ion transport through CFTR and measuring the resulting changes in transepithelial voltage (Vte) and current (ISC). While this technique is suitable for measuring the maximum functional capacity of CFTR, it may not provide an accurate estimate of in vivo CFTR activity. To test if CFTR-mediated ion transport could be measured in the absence of acute CFTR stimulation, primary airway epithelia were analyzed in an Ussing chamber with treatment of amiloride followed by CFTR(inh)-172 without acute activation of CFTR. Non-CF epithelia demonstrated a decrease in Vte and ISC following exposure to CFTR(inh)-172 and in the absence of forskolin/IBMX (F/I); this decrease is interpreted as a measure of spontaneous CFTR activity present in these epithelia. In F508del/F508del CFTR epithelia, F/I-induced changes in Vte and ISC were ~ fourfold increased after treatment with VX-809/VX-770, while the magnitude of spontaneous CFTR activities were only ~ 1.6-fold increased after VX-809/VX-770 treatment. Method-dependent discrepancies in the responses of other CF epithelia to modulator treatments were observed. These results serve as a proof of concept for the analysis of CFTR modulator responses in vitro in the absence of acute CFTR activation. Future studies will determine the usefulness of this approach in the development of novel CFTR modulator therapies.

Molecular Mechanisms of Renal Magnesium Reabsorption

Magnesium is an essential cofactor in many cellular processes, and aberrations in magnesium homeostasis can have life-threatening consequences. The kidney plays a central role in maintaining serum magnesium within a narrow range (0.70 to 1.10 mmol/L). Along the proximal tubule and thick ascending limbs, magnesium reabsorption occurs via paracellular pathways. Members of the claudin family form the magnesium pores in these segments, and also regulate magnesium reabsorption by adjusting the transepithelial voltage that drives it. Along the distal convoluted tubule transcellular reabsorption via heteromeric TRPM6/7 channels predominates, though paracellular reabsorption may also occur. In this segment, the NaCl cotransporter plays a critical role in determining transcellular magnesium reabsorption. While the general machinery involved in renal magnesium reabsorption has been identified by studying genetic forms of magnesium imbalance, the mechanisms regulating it are poorly understood. This review discusses pathways of renal magnesium reabsorption by different segments of the nephron, emphasizing newer findings that provide insight into regulatory process, and outlining critical unanswered questions.

ENaC inhibition stimulates HCl secretion in the mouse cortical collecting duct. I. Stilbene-sensitive Cl− secretion

Inhibition of the epithelial Na+ channel (ENaC) reduces Cl− absorption in cortical collecting ducts (CCDs) from aldosterone-treated rats and mice. Since ENaC does not transport Cl−, the purpose of the present study was to explore how ENaC modulates Cl− absorption in mouse CCDs perfused in vitro. Therefore, we measured transepithelial Cl− flux and transepithelial voltage in CCDs perfused in vitro taken from mice that consumed a NaCl-replete diet alone or the diet with aldosterone administered by minipump. We observed that application of an ENaC inhibitor [benzamil (3 μM)] to the luminal fluid unmasks conductive Cl− secretion. During ENaC blockade, this Cl− secretion fell with the application of a nonselective Cl− channel blocker [DIDS (100 μM)] to the perfusate. While single channel recordings of intercalated cell apical membranes in split-open CCDs demonstrated a Cl− channel with properties that resemble the ClC family of Cl− channels, ClC-5 is not the primary pathway for benzamil-sensitive Cl− flux. In conclusion, first, in CCDs from aldosterone-treated mice, most Cl− absorption is benzamil sensitive, and, second, benzamil application stimulates stilbene-sensitive conductive Cl− secretion, which occurs through a ClC-5-independent pathway.

ENaC inhibition stimulates HCl secretion in the mouse cortical collecting duct. II. Bafilomycin-sensitive H+ secretion

Epithelial Na+ channel (ENaC) blockade stimulates stilbene-sensitive conductive Cl− secretion in the mouse cortical collecting duct (CCD). This study's purpose was to determine the co-ion that accompanies benzamil- and DIDS-sensitive Cl− flux. Thus transepithelial voltage, VT, as well as total CO2 (tCO2) and Cl− flux were measured in CCDs from aldosterone-treated mice consuming a NaCl-replete diet. We reasoned that if stilbene inhibitors (DIDS) reduce conductive anion secretion they should reduce the lumen-negative VT. However, during ENaC blockade (benzamil, 3 μM), DIDS (100 μM) application to the perfusate reduced net H+ secretion, which increased the lumen-negative VT. Conversely, ENaC blockade alone stimulated H+ secretion, which reduced the lumen-negative VT. Application of an ENaC inhibitor to the perfusate reduced the lumen-negative VT, increased intercalated cell intracellular pH, and reduced net tCO2 secretion. However, benzamil did not change tCO2 flux during apical H+-ATPase blockade (bafilomycin, 5 nM). The increment in H+ secretion observed with benzamil application contributes to the fall in VT observed with application of this diuretic. As such, ENaC blockade reduces the lumen-negative VT by inhibiting conductive Na+ absorption and by stimulating H+ secretion by type A intercalated cells. In conclusion, 1) in CCDs from aldosterone-treated mice, benzamil application stimulates HCl secretion mediated by the apical H+-ATPase and a yet to be identified conductive Cl− transport pathway; 2) benzamil-induced HCl secretion is reversed with the application of stilbene inhibitors or H+-ATPase inhibitors to the perfusate; and 3) benzamil reduces VT not only by inhibiting conductive Na+ absorption, but also by stimulating H+ secretion.

Pendrin gene ablation alters ENaC subcellular distribution and open probability

The present study explored whether the intercalated cell Cl−/HCO3− exchanger pendrin modulates epithelial Na+ channel (ENaC) function by changing channel open probability and/or channel density. To do so, we measured ENaC subunit subcellular distribution by immunohistochemistry, single channel recordings in split open cortical collecting ducts (CCDs), as well as transepithelial voltage and Na+ absorption in CCDs from aldosterone-treated wild-type and pendrin-null mice. Because pendrin gene ablation reduced 70-kDa more than 85-kDa γ-ENaC band density, we asked if pendrin gene ablation interferes with ENaC cleavage. We observed that ENaC-cleaving protease application (trypsin) increased the lumen-negative transepithelial voltage in pendrin-null mice but not in wild-type mice, which raised the possibility that pendrin gene ablation blunts ENaC cleavage, thereby reducing open probability. In mice harboring wild-type ENaC, pendrin gene ablation reduced ENaC-mediated Na+ absorption by reducing channel open probability as well as by reducing channel density through changes in subunit total protein abundance and subcellular distribution. Further experiments used mice with blunted ENaC endocytosis and degradation (Liddle's syndrome) to explore the significance of pendrin-dependent changes in ENaC open probability. In mouse models of Liddle's syndrome, pendrin gene ablation did not change ENaC subunit total protein abundance, subcellular distribution, or channel density, but markedly reduced channel open probability. We conclude that in mice harboring wild-type ENaC, pendrin modulates ENaC function through changes in subunit abundance, subcellular distribution, and channel open probability. In a mouse model of Liddle's syndrome, however, pendrin gene ablation reduces channel activity mainly through changes in open probability.

Early-life dietary spray-dried plasma influences immunological and intestinal injury responses to later-lifeSalmonellatyphimuriumchallenge

Increasing evidence supports the concept that early-life environmental influences, including nutrition and stress, have an impact on long-term health outcomes and disease susceptibility. The objective of the present study was to determine whether dietary spray-dried plasma (SDP), fed during the first 2 weeks post-weaning (PW), influences subsequent immunological and intestinal injury responses toSalmonellatyphimuriumchallenge. A total of thirty-two piglets (age 16–17 d) were weaned onto nursery diets containing 0, 2·5 % SDP (fed for 7 d PW) or 5 % SDP (fed for 14 d PW), and were then fed control diets (without SDP), for the remainder of the experiment. At 34 d PW (age 50 d), pigs were challenged with 3 × 109colony-forming units ofS. typhimurium. A control group (non-challenged) that was fed 0 % SDP in the nursery was included. At 2 d post-challenge, the distal ileum was harvested for the measurement of inflammatory, histological and intestinal physiological parameters.S.typhimuriumchallenge induced elevated ileal histological scores, myeloperoxidase (MPO), IL-8 and TNF, and increased intestinal permeability (indicated by reduced transepithelial voltage (potential difference) and elevated 4 kDa fluorescein isothiocyanate dextran (FD4) flux rates). Compared withS.typhimurium-challenged controls (0 % SDP), pigs fed the 5 % SDP-14 d diet exhibited reduced ileal histological scores, MPO levels, IL-8 levels and FD4 flux rates. Pigs fed the 5 % SDP-14 d nursery diet exhibited increased levels of plasma and ileal TNF-α in response to the challenge, compared with the other treatments. These results indicate that inclusion of SDP in PW diets can have an influence on subsequent immunological and intestinal injury responses induced by later-lifeS.typhimuriumchallenge.

ENaC inhibition stimulates Cl− secretion in the mouse cortical collecting duct through an NKCC1-dependent mechanism

In cortical collecting ducts (CCDs) perfused in vitro, inhibiting the epithelial Na+ channel (ENaC) reduces Cl− absorption. Since ENaC does not transport Cl−, the purpose of this study was to determine how ENaC modulates Cl− absorption. Thus, Cl− absorption was measured in CCDs perfused in vitro that were taken from mice given aldosterone for 7 days. In wild-type mice, we observed no effect of luminal hydrochlorothiazide on either Cl− absorption or transepithelial voltage ( VT). However, application of an ENaC inhibitor [benzamil (3 μM)] to the luminal fluid or application of a Na+-K+-ATPase inhibitor to the bath reduced Cl− absorption by ∼66–75% and nearly obliterated lumen-negative VT. In contrast, ENaC inhibition had no effect in CCDs from collecting duct-specific ENaC-null mice (Hoxb7:CRE, Scnn1aloxlox). Whereas benzamil-sensitive Cl− absorption did not depend on CFTR, application of a Na+-K+-2Cl− cotransport inhibitor (bumetanide) to the bath or ablation of the gene encoding Na+-K+-2Cl− cotransporter 1 (NKCC1) blunted benzamil-sensitive Cl− absorption, although the benzamil-sensitive component of VT was unaffected. In conclusion, first, in CCDs from aldosterone-treated mice, most Cl− absorption is benzamil sensitive, whereas thiazide-sensitive Cl− absorption is undetectable. Second, benzamil-sensitive Cl− absorption occurs by inhibition of ENaC, possibly due to elimination of lumen-negative VT. Finally, benzamil-sensitive Cl− flux occurs, at least in part, through transcellular transport through a pathway that depends on NKCC1.

Interleukin-6 stimulates epithelial sodium channels in mouse cortical collecting duct cells

The aim of this study is to elucidate the effects of interleukin-6 (IL-6) on the expression and activity of the epithelial sodium channel (ENaC), which is one of the key mechanisms underlying tubular sodium reabsorption. M-1 cortical collecting duct cells were treated with IL-6 (100 ng/ml) for 12 h. Real-time polymerase chain reaction and immunoblotting were employed to examine the mRNA and protein abundance. Transepithelial voltage ( Vte) and resistance ( Rte) were measured with an ohm/voltmeter (EVOM, WPI). The equivalent current was calculated as the ratio of Vte to Rte. Treatment with IL-6 ( n = 5) increased the mRNA abundance of α-ENaC by 11 ± 7% ( P = not significant), β-ENaC by 78 ± 14% ( P = 0.01), γ-ENaC by 185 ± 38% ( P = 0.02), and prostasin by 29 ± 5% ( P = 0.01), all normalized by β-actin. Treatment with IL-6 increased the protein expression of α-ENaC by 19 ± 3% ( P = 0.001), β-ENaC by 89 ± 21% ( P = 0.01), γ-ENaC by 36 ± 12% ( P = 0.02), and prostasin by 33 ± 6% ( P = 0.02). The amiloride-sensitive sodium current increased by 37 ± 5%, from 6.0 ± 0.4 to 8.2 ± 0.3 μA/cm2 ( P < 0.01), in the cells treated with IL-6 compared with controls ( P = 0.01). Aprotinin (28 μg/ml), a prostasin inhibitor, reduced the amiloride-sensitive sodium current by 61 ± 5%, from 6.1 ± 0.3 to 3.7 ± 0.2 μA/cm2 ( P = 0.01). The magnitude of the IL-6-induced amiloride-sensitive sodium current in the presence of aprotinin dropped by 57 ± 2%, from 8.6 ± 0.2 to 4.9 ± 0.2 μA/cm2 ( P < 0.01). This study has identified a novel function of IL-6, namely, IL-6 may activate ENaC. Therefore, renal inflammation mediated by IL-6 likely contributes to impaired pressure natriuresis.