Inhibition of the sodium-dependent HCO3- transporter SLC4A4, produces a cystic fibrosis-like airway disease phenotype

  1. Vinciane Saint-Criq
  2. Anita Guequén
  3. Amber R Philp
  4. Sandra Villanueva
  5. Tábata Apablaza
  6. Ignacio Fernández-Moncada
  7. Agustín Mansilla
  8. Livia Delpiano
  9. Iván Ruminot
  10. Cristian Carrasco
  11. Michael A Gray
  12. Carlos A Flores  Is a corresponding author
  1. Biosciences Institute, The Medical School, Newcastle University, United Kingdom
  2. Centro de Estudios Científicos, Chile
  3. Universidad Austral de Chile, Chile
  4. Universidad San Sebastián, Chile
  5. Subdepartamento de Anatomía Patológica, Hospital Base de Valdivia, Chile

Abstract

Bicarbonate secretion is a fundamental process involved in maintaining acid-base homeostasis. Disruption of bicarbonate entry into airway lumen, as has been observed in cystic fibrosis, produces several defects in lung function due to thick mucus accumulation. Bicarbonate is critical for correct mucin deployment and there is increasing interest in understanding its role in airway physiology, particularly in the initiation of lung disease in children affected by cystic fibrosis, in the absence of detectable bacterial infection. The current model of anion secretion in mammalian airways consists of CFTR and TMEM16A as apical anion exit channels, with limited capacity for bicarbonate transport compared to chloride. However, both channels can couple to SLC26A4 anion exchanger to maximise bicarbonate secretion. Nevertheless, current models lack any details about the identity of the basolateral protein(s) responsible for bicarbonate uptake into airway epithelial cells. We report herein that the electrogenic, sodium-dependent, bicarbonate cotransporter, SLC4A4, is expressed in the basolateral membrane of human and mouse airways, and that it’s pharmacological inhibition or genetic silencing reduces bicarbonate secretion. In fully differentiated primary human airway cells cultures, SLC4A4 inhibition induced an acidification of the airways surface liquid and markedly reduced the capacity of cells to recover from an acid load. Studies in the Slc4a4-null mice revealed a previously unreported lung phenotype, characterized by mucus accumulation and reduced mucociliary clearance. Collectively, our results demonstrate that the reduction of SLC4A4 function induced a CF-like phenotype, even when chloride secretion remained intact, highlighting the important role SLC4A4 plays in bicarbonate secretion and mammalian airway function.

Editor's evaluation

This paper is of interest to scientists and clinicians within the field of muco-obstructive diseases in the airways, such as cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD). It identifies the sodium-bicarbonate cotransporter SLC4A4 as a key component of the mechanism by which normal airways prevent the formation of sticky mucus and defend their selves against bacterial and viral infections.

https://doi.org/10.7554/eLife.75871.sa0

Introduction

Bicarbonate (HCO3-) and chloride (Cl-) are actively secreted into the lumen of airways by the lining epithelial cells. Even though, the pathophysiological consequences of decreased secretion of these anions has been extensively documented in cystic fibrosis (CF), the most common, autosomal recessive, disease in humans, there is still much discussion whether HCO3- secretion per se affects airway homeostasis (Zajac et al., 2021; Morrison et al., 2022). Impaired Cl- secretion reduces the volume of the fluid that covers the airway epithelium, the airway surface liquid (ASL), leading to ciliary dysfunction and promoting mucus stasis and airway obstruction (Cantin et al., 2006; Saint-Criq and Gray, 2017). Deficient HCO3- secretion reduces ASL pH which compromises post-secretory mucin maturation and clearance (Hoegger et al., 2014), impairs the antimicrobial function of epithelial cells (Pezzulo et al., 2012; Shah et al., 2016) and increases fluid absorption, further decreasing ASL hydration (Garland et al., 2013). Whilst direct measurement of pH in the distal airways of CF children showed no acidification of the ASL (Schultz et al., 2017), it has been demonstrated that the addition of HCO3- induced an increase in ASL height in human airway epithelial cells (hAECs) cultures and restored the normal properties of mucus from CF patients (Garland et al., 2013; Stigliani et al., 2016). Moreover, the use of aerosolized HCO3- into CF-pig airways increased bacterial killing, clearly indicating that HCO3- supplementation can correct inherent defects of CF in the airways (Pezzulo et al., 2012; Kim et al., 2021).

Even though, impaired HCO3- secretion is recognised as a detrimental component of CF airway disease, a full mechanistic understanding of the process and players involved in transcellular HCO3- transport in the airways is lacking. In normal airways, HCO3- is secreted by CFTR and TMEM16A channels, but after inflammatory signalling, HCO3- secretion is augmented through increased expression of the Cl- /HCO3- exchanger Pendrin (SLC26A4) (Han et al., 2016; Poulsen et al., 1994; Kim et al., 2019; Rehman et al., 2020; Garnett et al., 2011). Importantly, the mechanism of basolateral HCO3- transport/uptake in native pulmonary tissues is still lacking. We reasoned that the identification and functional inhibition of such basolateral membrane proteins could be used as a proof-of-concept to better understand the role of HCO3- secretion in airway homeostasis without altering Cl- secretion. Here using in vitro fully differentiated human bronchial epithelial cells, we identified the Na+-dependent, HCO3- electrogenic cotransporter (NBCe1), SLC4A4, as the basolateral protein responsible for HCO3- influx, which couples to apical CFTR for HCO3- secretion into the ASL. Importantly, SLC4A4 inhibition induced acidification of the ASL, revealing the pivotal role of this cotransporter in airway pH homeostasis. These observations were further tested in murine models, and revealed that SLC4A4 is involved in both basal and Ca2+-stimulated HCO3- secretion in mice airways. Strikingly, an Slc4a4-/- mouse model showed significant pathological signs of muco-obstructive disease and reduced mucociliary clearance, confirming that inhibition of HCO3- secretion alters airway homeostasis, mimicking what has been observed in human CF, and identifying a critical role played by SLC4A4.

Results

Primary human airway epithelial cells express bicarbonate transporters of the SLC4A family

We first investigated, by PCR, whether primary hAECs expressed different members of the SLC4A family of Na+-coupled HCO3- transporters (NCBT) and isolated RNA from kidney and the Calu-3 cell line, which was derived from a metastatic site of a lung adenocarcinoma, as positive controls. The use of specific primers (Supplementary file 1) for each NCBT revealed that SLC4A4, SLC4A5, SLC4A7, and SLC4A8 are expressed at mRNA levels in primary hAECs from three different individuals (P1, P2, P3, Figure 1—figure supplement 1A-E). SLC4A10 showed near undetectable level of mRNA expression (Figure 1—figure supplement 1F). Interestingly, isoforms B/C of NBCe1 (known as the pancreatic isoform, Figure 1—figure supplement 1A) were more highly expressed than isoform A (known as the kidney isoform; Figure 1—figure supplement 1E). Data extraction from previously published RNA-seq results (Saint-Criq et al., 2020) (GEO series accession number GSE154905) confirmed the pattern of expression of these isoforms (Figure 1—figure supplement 1G) and revealed that SLC4A4 is the most expressed member, closely followed by SLC4A7, SLC4A8, and SLC4A5. Other candidate HCO3- transporters such as Bestrophins were also expressed in these cells but with one to two logs lower than SLC4A4, SLC4A7, and SLC4A8 (Figure 1—figure supplement 1G).

SLC4A4 is central for bicarbonate secretion and intracellular pH homeostasis in human airway cells

We then tested whether there was an active NCBT under unstimulated and stimulated conditions in primary hAECs. The cell cultures were mounted in Ussing chambers in buffers containing either HCO3- (but no Cl-) or HEPES (no HCO3-), and treated basolaterally with the inhibitor S0859 (30 μM) followed by Forskolin (Fsk; 10 µM). Results, shown in Figure 1 confirmed that, in the absence of stimulants, HCO3- secretion was inhibited by S0859 (Figure 1A and B) and that this pharmacological inhibitor did not have any effect on short-circuit current (Isc) in the absence of HCO3- (Figure 1D and E). Interestingly, S0859 did not reduce Fsk-stimulated HCO3- secretion (Figure 1C) under these conditions. These results show that there is an electrogenic HCO3- transporter at the basolateral membrane of hAECs, which is consistent with SLC4A4, since SLC4A7 and SLC4A8 are electroneutral (Parker and Boron, 2013), and SLC4A5 has been shown to be localized to the apical membrane of renal epithelial cells and cholangiocytes (Gildea et al., 2015; Abuladze et al., 1998). Next, we used intracellular pH (pHi) measurements to functionally investigate NBCe1 activity using a CO2-induced acidification protocol (Theparambil et al., 2015). As shown in Figure 1E, exposing cells bilaterally to a HCO3-/CO2-gassed KRB solution induced a transient acidification. On the other hand, an apical-only CO2 exposure, in the absence of basolateral HCO3-, induced a sustained acidification (of the same amplitude as with bilateral HCO3-/CO2, Figure 1F and H), that recovered when HCO3- was re-introduced basolaterally (Figure 1F,I). This pHi recovery depended on the presence of Na+ in the basolateral solution (Figure 1G) consistent with a Na+-coupled HCO3- transporter, which was confirmed using S0859 which blocked the pHi recovery from the CO2-induced acidification. In order to isolate NBCe1-dependent changes in pHi, the contribution of Na+/H+ exchanger NHE was inhibited using 100 μM Dimethyl amiloride (DMA). In this condition, S0859 still significantly decreased the rate of pHi recovery from the CO2-induced acidification (Figure 1J and K).

Figure 1 with 1 supplement see all
Basal bicarbonate secretion requires SLC4A4 activity in primary hAECs.

Mean traces (+ standard error of the mean) of Isc in (A) Cl- free solution (n=14) and (D) HCO3- free solution (n=10) of primary hAECs cultures (dotted lines represent the time of addition of drugs). Summary of S0859-induced changes in Isc in the presence (B) or absence (E) of HCO3- in primary hAECs cultures and Fsk-induced HCO3- secretion (C) in Cl- free buffer (on panels B, C,, and E, each dot represents an independent experiment; means ± sem are shown next to the individual data; respectively n=17, n=14, and n=11 independent experiments using cells from N=3 donors; two-tailed, paired t-test). (F) Representative pHi trace of primary hAECs bathed first in bilateral HCO3- KRB (gassed with 5% CO2) then bilateral Hepes buffered KRB (no HCO3-, no CO2) and then bilateral HCO3- KRB (gassed with 5% CO2). CO2 removal and re-introduction is marked by a transient increase and decrease in pHi respectively. (G) Representative trace of pHi recovery after CO2-induced acidification in the absence of basolateral HCO3-. (H) Representative trace of pHi recovery after CO2-induced acidification in the absence of basolateral Na+ and HCO3-. (I) Summary of the magnitude of CO2-induced acidification (bilateral bicarbonate, n=7, apical bicarbonate n=14, unpaired t-test, bars represent mean ± standard deviation (S.D.)). (J) Mean percentage of pHi recovery after perfusion of the different solutions (Bilateral Bicarbonate, n=7; Apical Bicarbonate, n=14; Bilateral Bicarbonate after Apical Bicarbonate-, n=5; Na+-free buffer, n=8; Bilateral Bicarbonate after Na+-free buffer, n=8; One-way ANOVA with Holm-Sidak correction for multiple comparison tests, bar graph represents mean ± S.D.). (K) Representative trace of intracellular pH measurements showing recovery from CO2-induced acidification in the presence (red line) or absence (blue line) of NBC inhibitor S0859. (L) Summary of rates of recovery from CO2-induced acidification in the presence of NHE inhibitor (Dimethyl Amiloride, DMA, 100 μM) and in the presence (red bar) or absence (blue bar) of S0859 (30 μM), (n=3, paired t-test; bars represent mean ± S.D.).

Basolateral HCO3- uptake is essential for ASL pH homeostasis

As Ussing chamber and pHi experiments use standard buffer solutions that are unlikely to fully resemble that of the ASL, we then used fully differentiated hAECs at the air-liquid interface to establish whether SLC4A4 is involved in airway epithelial pH homeostasis in a setting that is more physiologically relevant. In order to test whether HCO3- transport by basolateral SLC4A4 impacted apical HCO3- secretion under thin film conditions, we measured the effect of S0859 on ASL pH. First, S0859 was added basolaterally to primary hAECs and ASL pH continuously measured. NBCe1 inhibition significantly decreased ASL pH under resting conditions with a t1/2 of 46 min (Figure 2A,B), and partially prevented the Fsk-induced increase in ASL pH which we have previously shown was via CFTR (Delpiano et al., 2018; Saint-Criq et al., 2019; Figure 2C,D), increasing the half-time response to Fsk from 25 to 31 min. Moreover, when S0859 was added after Fsk, it significantly reduced the Fsk-induced, CFTR-dependent, increase in ASL pH with a half-time of 14 min (Figure 2E,F) confirming the central role of SLC4A4 cotransporter in ASL pH homeostasis under both resting and stimulated conditions. It is worth noting that the changes in ASL pH were much slower than in the pHi experiments, which can be explained by the differences in technical (non-perfused ASL pH versus bilaterally perfused pHi) and experimental conditions set-ups (chemical induced versus. CO2-induced changes in pH).

S0859 decreases resting ASL pH in primary human airway epithelial cells (hAECs), by blocking basolateral SLC4A4.

(A) Mean (+ S.E.M.) trace of ASL pH recordings. ASL pH was measured under resting conditions for 1.5 hr before S0859 (30 µM) was added basolaterally. (B) Mean resting ASL pH before (black circles) and after (blue circles) addition of basolateral S0859 (n=9 independent experiments performed on epithelial cells from N=3 donors; paired t-test). (C) Mean (+ SEM) traces of ASL pH from hAECs pre-treated for 3 h with vehicle control (DMSO, black trace) or S0859 (30 µM, basolateral, blue trace). (D) Mean Forskolin (Fsk)-induced changes in ASL pH in hAECs treated with DMSO (black circles) or S0859 (blue circles) (n=9 independent experiments performed on epithelial cells from N=3 donors; paired t-test). (E) Mean (+ SEM) traces of ASL pH from hAECs treated with Fsk for 2.5 hr and then S0859. (F) Mean Fsk-stimulated ASL pH before (black circles) and after (blue circles) addition of basolateral S0859 (n=9 independent experiments performed on epithelial cells from N=3 donors; paired t-test). (G–H) SLC4A4 locates in the basolateral membrane of acetylated tubulin (Ac-tubulin) positive human airway epithelial cells. (I–J) correspond to negative controls for anti-SLC4A4 omitted antibody. Representative images of three different samples. Bar 20 µM.

Finally, immunolocalization of SLC4A4 protein in human airway tissues showed intracellular and basolateral membrane staining in epithelial cells that were also positively stained for acetylated-tubulin indicating that SLC4A4 is preferentially expressed in ciliated cells in human airways (Figure 2G,H).

Bicarbonate secretion is calcium-activated in mouse trachea

To investigate the expression of SLC4 exchangers in mouse airway epithelium, we performed RT-PCR of epithelial cells from mouse tracheas and observed that several members of the SLC4 family including Slc4a4, Slc4a5, Slc4a7, and Slc4a10 were expressed (Figure 3—figure supplement 1 A-E). Studies of Slc4a4 isoforms demonstrated that isoform B/C but not isoform A was expressed in mouse airways (Figure 3—figure supplement 1F). Next we characterized HCO3- secretion in the mouse. Ussing chamber experiments performed in freshly excised mouse trachea using HCO3- (Figure 3A) or HEPES (Figure 3B) buffered solutions, showed that UTP-induced an anion current that was significantly reduced in the absence of HCO3- (–138±28 to -82±15 µA cm–2; p<0.01 Mann-Whitney test), but no significant effect on the cAMP-induced anion secretion, or the amiloride-sensitive sodium absorption (Figure 3C) was detected as previously shown (Anagnostopoulou et al., 2012). Complementary studies in HCO3- buffer showed that de novo synthesis of HCO3- was not participating in the UTP-induced electrogenic anion secretion, as incubation of tracheas with acetazolamide didn’t affect the magnitude of the UTP-induced current (–124±14 µA cm–2; p>0.05 One-way ANOVA).

Figure 3 with 2 supplements see all
Basal and inducible bicarbonate secretion relies in SLC4A4 activity in mouse tracheal epithelium.

Representative traces of ISC in (A) bicarbonate and (B) HEPES buffer of freshly excised mouse tracheas. (C) Summary of ΔISC values for amiloride-sensitive Na+ absorption, IBMX +Fsk-induced and A.U.C. ISC UTP-induced anion secretion in mouse tracheas; n=6 for each condition; Mann-Whitney Rank Sum Test. Representative ISC traces of S0859 effect on unstimulated tracheas in (D) bicarbonate and (E) HEPES buffer. (F) Summary of ΔISC values for S0859 effect, n=5 for each condition; Mann-Whitney Rank Sum Test. Representative ISC traces for UTP-induced anion secretion in absence (G and I) or presence (H and J) of 30 µM S0859 in buffer bicarbonate (G and H) or HEPES (I and J). All in the presence of 10 µM amiloride. (K) Summary of experiments as presented in G-J; n=5 but HEPES + S0859 n=6; ANOVA on Ranks. Bars are mean ± S.E.M. (L) Average response of determination of pHi in epithelial cells isolated from wild type mouse trachea and loaded with BCECF that were stimulated with 100 µM UTP and switched to 12.5 mM bicarbonate buffered solution. Experiments performed in bicarbonate buffer in blue and HEPES in red. (M) Summary of ΔpHi from experiments as those in (L) including a data set of cells maintained in HEPES buffer; Middle line of the box plot indicates the median; n=15 cells from four different mice, n=11 cells, three different mice and n=13 cells, three different mice respectively; ANOVA on Ranks.

Using the SLC4A4 blocker S0859, we observed the inhibition of the cAMP-induced anion current (ΔIsc –12.2±2.4 µA cm–2) suggesting that SLC4A4 might participate in the cAMP-response (Figure 3—figure supplement 2A-C). Nevertheless, when the TMEM16A/CFTR inhibitor, CaCCinhA01, was used to block the cAMP-induced current, further addition of S0859 was still able to induce a reduction in the current and of similar magnitude as shown in Figure 3—figure supplement 2A (Figure 3—figure supplement 2D-F; –10.1±2.8 µA cm–2), confirming that electrogenic-bicarbonate secretion was not significantly stimulated by cAMP, indicating that basal HCO3- secretion occurs in mouse trachea. To confirm this last hypothesis we added S0589 to tissues pre-incubated with amiloride and observed a reduction in the basal current only in HCO3- buffer (–13.9±3.3 to -3.0±1.6 Δ µA cm–2 for HCO3- vs HEPES buffer; p<0.02; Mann-Whitney; Figure 3D–F). The magnitude of basal HCO3- secretion inhibited by S0859 was similar to the experiments summarized in Figure 3—figure supplement 2C and F. Of note, the addition of S0859 to the tracheas induced a fast and transient negative change in Isc as observed in Figure 3D and E and Figure 3—figure supplement 2A and D, that has been also observed in human cells (Gorrieri et al., 2016), and that might be due to off-targets of the blocker like other SLC4A or SLC16A transporters as previously described (Heidtmann et al., 2015; Schwab et al., 2005; Ch’en et al., 2008).

To further characterize the Ca2+-activated anion secretion, the UTP response was tested with no involvement of cAMP-induced secretion. As can be observed (Figure 3G and H) the UTP-induced anion secretion in tracheas maintained in HCO3- buffer was significantly reduced by previous addition of S0859 (–368±25 to -200±17 µA cm–2; p<0.001 One-way ANOVA). The UTP response was also reduced when HCO3- was replaced with HEPES buffer (Figure 3I) (–199±25 µA cm–2; p<0.001 One-way ANOVA), but the addition of S0859 induced no significant reduction of the UTP-induced anion secretion in tissues maintained in HEPES buffer (Figure 3J) (–142±12 µA cm–2; p>0.05 One-way ANOVA).

SLC4A4 participates in intracellular pH homeostasis in mouse airway epithelial cells

We reasoned that the UTP-induced HCO3- exit would lead to cytoplasmic acidification and therefore we monitored intracellular pH of BCECF-loaded murine airway cells. As shown in Figure 3L, UTP induced an intracellular acidification (ΔpHi –0.25±0.02) that was significantly reduced when cells were placed in low Cl- buffer (ΔpHi –0.11±0.02), indicating the existence of Cl-/HCO3- exchange. Figure 3M summarizes changes in intracellular pH and includes experiments in HEPES buffer, which shows that UTP was almost unable to induce intracellular acidification (ΔpHi –0.01±0.01) in absence of HCO3-. To test if the UTP-induced intracellular acidification was dependent on SLC4A4 activity, we tested the S0859 inhibitor and observed acidification of the intracellular compartment and prevention of UTP-induced acidification (Figure 3—figure supplement 2G-H). Washout of S0859 partially restored pHi and UTP-induced acidification. (Figure 3—figure supplement 2G-H; –0.05±0.01 to -0.13±0.03 ΔpHi, for UTP with S0559 and UTP post washout, respectively; p>0.002; Mann-Whitney). These data suggest that both basal and UTP-induced HCO3- secretion are dependent on SLC4A4 activity in mouse airway epithelial cells.

The genetic inactivation of Slc4a4 induces a cystic fibrosis-like phenotype in mouse airways

As explained in the methods section, we decided to work with wild type and Slc4a4-/- on the hybrid background, at 16–20 days of age. First, we observed that Slc4a4-/- animals were affected by defects in tracheal cartilage formation with the presence of ventral gaps and abnormal patterns on the rostrocaudal side (Figure 4A). In wild-type animals, immunolocalization of SLC4A4 showed strong localization in the airway epithelium but the signal was nearly absent in the airways from the Slc4a4-/- mice (Figure 4B). Using the same antibody, we showed that SLC4A4 was preferentially expressed in CCSP-positive cells that correspond to Club cells and was excluded from cells positive to acetylated-Tubulin, that identify ciliated cells (Figure 4C). This pattern of expression was maintained in distal airway bronchi and bronchioles (Figure 4—figure supplement 1C). Further histological examination of the Slc4a4-/- mouse airways demonstrated the presence of adherent mucus at the surface of the tracheal epithelium (Figure 4D and Figure 4—figure supplement 1C,D) as well as in the bronchi (Figure 4—figure supplement 1E-G) and bronchioli (Figure 4—figure supplement 1H). Signs of damaged epithelium was also observed as interruptions in the epithelial layer facing the lumen in the Slc4a4-/- airways (Figure 4D and Figure 4—figure supplement 1F,H), that might explain the decreased Rte of the tracheas placed in Ussing chambers and that prevented electrophysiological measurements in the Slc4a4-/- tracheas (Figure 4E).

Figure 4 with 1 supplement see all
The Slc4a4-/- bear signs of muco-obstructive airway disease.

(A) Ventral view of Alcian blue stained tracheas of 17 days old mice. Red arrow heads show incomplete cartilage rings in the Slc4a4-/- mouse; representative image of 3 animals per genotype. (B) SLC4A4 staining of epithelial cells is absent in the Scl4a4-/- lung tissues; representative images of three different animals per genotype. (C) Epithelial SLC4A4 co-localizes with CCSP; representative images of three different animals. (D) Mucin staining showing mucus adhered to the epithelial surface and epithelial damage (red arrow heads); representative images of five animals per genotype. (E) Summary of Rte values for wild type and Scl4a4-/- tracheas; dashed line indicates 50 Ωcm–2; n=8 for wild types and n=6 for Scl4a4-/- tracheas. (F) Representative traces of pHi in airway cells from wild type (blue) and Slc4a4-/- (red) animals and (G) summary of UTP-inducedΔpHi including mean ± S.E.M. and individual cells; n=8 cells for wild type and n=6 cells for Scl4a4-/-, three different animals per genotype; Mann-Whitney Rank Sum Test. (H) Beads tracking of MCC experiments for wild type and Slc4a4-/- tracheas bathed with basolateral bicarbonate or HEPES buffer. Radius of the polar plots is 50 µm. Summary of MCC experiments for (I) speed, (J) total distance covered by beads. Bars indicate mean ± S.E.M.; n=5 for each genotype in bicarbonate buffer and n=4 for each genotype in HEPES buffer; ANOVA on ranks.

Genetic silencing of Slc4a4 impairs intracellular pH homeostasis and mucociliary clearance in mouse airways

In order to validate the role of SLC4A4 in pH homeostasis of murine airways, UTP-induced intracellular acidification was studied in airway cells isolated from the Slc4a4-/- mice. We observed a decrease in the magnitude of intracellular acidification in Slc4a4-/- cells when compared to those from wild type animals during UTP stimulation (–0.24±0.01 to -0.14±0.01 ΔpH[i], for wild type vs Slc4a4-/-) suggesting that an important amount of HCO3- accumulates in airways cells via SLC4A4 (Figure 4F–G). We also noticed that after UTP wash-out, the acidification persisted in the wild type cells but not in the Slc4a4-/- (Figure 4—figure supplement 1J; –0.09±0.02 to -0.02±0.01 ΔpH[i], for wild type vs Slc4a4-/-), suggesting that HCO3- secretion was sustained by SLC4A4 activity. Clearance of plastic beads, as a way to measure MCC, was studied in freshly isolated mouse tracheas whose mucosal side was exposed to air. As shown in the polar plots in Figure 4H the plastic beads covered a larger distance in wild type tissues and, in some cases, retrograde movement of beads was observed in the Slc4a4-/- trachea. A similar reduction in distance travelled was observed in wild type tissues bathed in HEPES buffer. The speed of plastic bead movement and total distance covered are summarized in Figure 4I and H. The use of HEPES buffer in the Slc4a4-/- tracheas showed no further effect on both speed and total distance travelled by the beads. This data set demonstrates that mucociliary clearance is significantly decreased when HCO3- transport is impaired after Slc4a4 silencing.

Discussion

SLC4A4 is a critical component of the bicarbonate secretory machinery

In this study, we have established that the Na+-coupled HCO3- transporter SLC4A4 or NBCe1 is central for bicarbonate transport, coupling to apical proteins to efficiently deliver bicarbonate in human and mouse airways. As the specificity of S0859 for SLC4A4 has been discussed and is still unclear (Heidtmann et al., 2015; Schwab et al., 2005; Ch’en et al., 2008), it is uncertain from our data whether SLC4A4 is the main and only actor regulating bicarbonate uptake in primary hAECs. However its importance in airway pH homeostasis is supported by strong evidence from the use of knock-out animals. Early characterization in canine and human airway epithelium indicated that HCO3- secretion occurs via CFTR, is activated by cAMP and dependent on basolateral Na+ Smith and Welsh, 1992, similar features were observed in the Calu-3 cell line (Devor et al., 1999). Our experiments in hAECs corroborated the cAMP-activation and Na+-dependence, that has also been observed by others in the same (Kim et al., 2019; Gorrieri et al., 2016) or other cell types (Kurtz, 2014). Nevertheless, and in contrast to human airway epithelium, our present data shows that HCO3- secretion in the mouse airways is mostly Ca+2- rather than cAMP-activated, but both human and mouse share a basal secretory component for HCO3- previously observed in human bronchioles (Shamsuddin and Quinton, 2019), and that we now show is dependent on SLC4A4 activity.

Basolateral localization of SLC4A4 was previously demonstrated in the Calu-3 cell line (Kreindler et al., 2006) but up to the date there has been no other studies investigating SLC4A4 localization in native airway tissues. Here, we demonstrate that human airways express SLC4A4 in ciliated cells preferentially, and that the observed basolateral localization correlates with the functional evidence provided, reinforcing the role SLC4A4 in basolateral HCO3- transport into the cells. Nevertheless, it must be considered that the sole expression of SLC4A4 does not assure HCO3- secretion as compelling evidence has demonstrated that apical CFTR is also necessary. While the calcium-activated TMEM16A channel has been shown to be expressed in goblet cells (Scudieri et al., 2012), both HCO3- transporters, CFTR and pendrin, have been localized in the apical membrane of ciliated hAECs cells supporting our observation (Kim et al., 2019; Kreda et al., 2005). Moreover, it has been demonstrated that SLC4A4-B, that we detected in human and mouse, and not the isoform A, is functionally coupled to CFTR through the IRBIT protein in pancreatic ducts (Yang et al., 2009; Shirakabe et al., 2006), further supporting the idea that a functional coupling in the same cell type is necessary for efficient HCO3- secretion. Even though, CFTR and Pendrin expression increases in secretory cells after cytokine stimulation, SLC4A4 remained unaltered, suggesting that other basolateral HCO3- transporter support HCO3- secretion in human secretory cells during the inflammatory response (Rehman et al., 2020). Finally, CFTR distribution in airway epithelium has been under renewed scrutiny after the recent discovery of the pulmonary ionocyte, a rare airway epithelial cell type that contains the highest amount of CFTR/Cftr transcripts in human and mouse (Montoro et al., 2018; Plasschaert et al., 2018). Nevertheless, recent and detailed studies demonstrate that most human airway epithelial cell types express CFTR including ciliated cells as was initially demonstrated (Engelhardt et al., 1994; Okuda et al., 2021).

We noticed that SLC4A4 localization in mouse was different from human airway epithelium, as the mouse SLC4A4 was expressed in CCSP +and not ciliated cells. Previously, CFTR and TMEM16A channels were specifically located in non-ciliated cells of mouse airways (Hahn et al., 2018), a distribution also conserved in the rat (Hahn et al., 2017). Pendrin, was also detected in non-ciliated secretory MUC5AC + cells, suggesting that a functional coupling for HCO3- secretion occurs in secretory non-ciliated cells of the mouse airways (Jia et al., 2016). Such a difference in expression of transporter proteins among human and mouse airways has frequently been described; for example CFTR, is not the principal Cl- transporter in the mouse airways and consequently silencing or mutation of Cftr in mice did not produce CF disease of the lungs (Ratcliff et al., 1993; Snouwaert et al., 1992). Furthermore, the expression in human and not mouse airways of another protein that influence ASL pH, the ATP12A H+/K+ ATPase, magnifies ASL acidification in human airways in CF disease (Shah et al., 2016).

Impaired bicarbonate secretion produces a CF-like phenotype in the mouse airways

We demonstrated that SLC4A4 activity is pivotal for HCO3- secretion. Inhibition of SLC4A4 induced acidification of the ASL in hAECs, an observation that suggests altering HCO3- delivery can initiate a CF-like phenotype, and that was confirmed in the Slc4a4-/- mouse model. Previous observations obtained by inducing silencing of Tmem16a in the mouse, or using cells carrying natural mutations in CFTR, affected both Cl- and HCO3- secretion making it difficult to understand what the consequences of reduced transport for each anion alone are (Gorrieri et al., 2016; Rock et al., 2009). As observed here, the silencing of Slcl4a4 induced a muco-obstructive phenotype in the mouse, whereas the silencing of Slc12a2, which encodes the bumetanide-sensitive NKCC1 co-transporter, which is essential for Cl- accumulation and secretion, did not (Grubb et al., 2001), suggesting that lack of HCO3- is pathologically more relevant than Cl- secretion in mouse airways. Indeed, a significant amount of experimental evidence, using different models and species, is consistent with our findings in the Slc4a4-/- mouse. For example, hAEC from CF patients produce acidic ASL and mucus that is more viscous than in cells from non-CF donors (Tang et al., 2016), the acidification of the ASL induced abnormal epithelial immune responses and reduced MCC that could be reversed after HCO3- supplementation (Pezzulo et al., 2012; Shah et al., 2016; Ferrera et al., 2021; Simonin et al., 2019; Birket et al., 2018; Cooper et al., 2013). Furthermore, HCO3- secretion is necessary for proper mucus release from hAECs (Gorrieri et al., 2016), and maintenance of normal amounts of ENaC-mediated Na+ absorption and ASL volume (Garland et al., 2013), functions that become abnormal due to acidic ASL pH. Even though technically challenging issues prevented us from measuring ASL pH in the mouse trachea, the reduction of HCO3- transport demonstrated here after SLC4A4 inhibition is consistent with the expected muco-obstructive phenotype, including reduced MCC and mucus accumulation.

Extra-renal phenotypes after Slc4a4 inactivation and human mutations

Mouse models of Slc4a4 silencing have shown differences in phenotypes depending on the isoforms affected. The Slc4a4-null animal, corresponding to the one used in the present studies, and the Slc4a4W516X/W516X avatar mouse engineered to mimic a non-sense mutation found in a human patient, are affected by severe metabolic acidosis due to proximal renal tubular acidosis (pRTA; Gawenis et al., 2007; Lo et al., 2011). Even though, decreased plasma HCO3- is observed in the Slc4a4-/- mice (5.3±0.5 mM; Gawenis et al., 2007), is unlikely that reduced HCO3- availability influenced mucus accumulation in the airways, as we have previously determined that the transporter is fully saturated at 3 mM HCO3- (Theparambil et al., 2014).

Extra-renal manifestations of Slc4a4 silencing in the mouse include growth retardation, ocular band keratopathy, splenomegaly, abnormal dentition and intestinal obstructions, most of which mimics the clinical findings observed in human patients. Nevertheless lung defects have not been reported in human patients, and observation which could be explained by the fact that human disease is milder than in mouse models. This might be related to the fact that at birth human kidneys are functionally more mature than in mouse (Takahashi et al., 2000) and for example, while heterozygous animals present mild pRTA, human patients bearing heterozygous SLC4A4 mutations did not show any signs of disease (Gawenis et al., 2007; Lo et al., 2011). Even though, a compensatory activity of other Na+-coupled HCO3- cotransporters, or exchangers, has been discarded in mouse, this possibility has not been examined in humans where compensatory activity might benefit patient’s health (Lo et al., 2011).

Both the Slc4a4-/- and W516X-mutant animals die soon after weaning as we also observed, but while the specific knock-out mouse for SLC4A4 isoform A (NBCe1-A) has a normal life span, the knock-out mouse for SLC4A4 isoforms B and C (NBCe1-B/C) is still lethal. This suggests that the cause of death of the animals was not due to metabolic acidosis, but rather due to extra renal phenotypes worsened by pRTA (Salerno et al., 2019; Lee et al., 2018). This observation might also be linked to the influence of modifier genes as observed in human patients and CF mouse models (Rozmahel et al., 1996; Cutting, 2010; Philp et al., 2018). In this regard, our observation that lethality is dependent on the genetic background of the animals supports such a possibility.

It will be of interest to use the Slc4a4 animal models and generate an airway specific null mouse to study the pathogenesis of muco-obstructive diseases of the lungs due to reduced HCO3- secretion. We believe that the elucidation of the transport systems that participate in pH maintenance in the airways offers the chance of increasing our current knowledge of the impact of impaired bicarbonate transport during health and disease.

Materials and methods

In vitro human airway epithelial cells studies

Cell culture

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Primary hAECs were a kind gift from Dr. Scott H. Randell (Marsico Lung Institute, The University of North Carolina at Chapel Hill, United States). The cells were obtained under protocol #03–1396 approved by the University of North Carolina at Chapel Hill Biomedical Institutional Review Board. Cells were expanded using the conditionally reprogrammed cell (CRC) culture method as previously described (Suprynowicz et al., 2012). Briefly, cells were seeded on 3T3J2 fibroblasts inactivated with mitomycin C (4 mg/ml, 2 hr, 37 °C) and grown in medium containing the ROCK inhibitor Y-27632 (10 mM, Tocris Biotechne, #1254) until they reached 80% confluence. Cells then underwent double trypsinization to remove the fibroblasts first and then detach the hAECs from the P150 dish. At that stage, cells were counted and could be frozen down. Cryopreserved cells were seeded onto semi-permeable supports (6.5 or 12 mm) in bilateral differentiating medium (ALI medium) as previously described (Randell et al., 2011). The apical medium was removed after 3–4 days and cells were then allowed to differentiate under air-liquid interface (ALI) conditions. Ciliogenesis started approximately 12–15 days after seeding and cells were used for experiments between days 25 and 35 after seeding.

Intracellular pH measurements

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Primary airway epithelial cells, grown on 12 mm Transwell inserts, were loaded with the pH-sensitive, fluorescent dye BCECF-AM (10 μM, ThemoFisher Scientific #B-1150) for 1 hr in a Na-HEPES buffered solution (130 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM Na-HEPES, and 5 mM D-glucose set to pH 7.4) at 37 °C. Cells were mounted on to the stage of a Nikon fluor inverted microscope and perfused with a modified Krebs (KRB) solution (115 mM NaCl, 5 mM KCl, 25 mM NaHCO3, 1 mM MgCl2, 1 mM CaCl2, and 5 mM D-glucose) gassed with 5% (v/v) CO2/95% (v/v) O2 or with a Na-HEPES-buffered solution gassed with 100% O2. Solutions were perfused across the apical and basolateral membranes at 37 °C at a speed of 3 ml min−1 and 6 ml min−1, respectively. To test the sodium dependence of bicarbonate transport, a Na+-free KRB solution was used in which 115 mM NMDG-Cl replaced NaCl, and 25 mM choline-HCO3 replaced NaHCO3. To measure the effect of NBC inhibition on the recovery from CO2 -induced acidification, epithelial cells were perfused basolaterally with 100 µM DMA (dimethyl amiloride, Sigma-Aldrich #A4562) to inhibit sodium-dependent hydrogen exchangers (NHEs) and 30 µM S0859 (Sigma-Aldrich #SML0638) to inhibit NBC. Intracellular pH (pHi) was measured using a Life Sciences Microfluorimeter System in which cells were alternately excited at 490 and 440 nm wavelengths every 1.024 s with emitted light collected at 510 nm. The ratio of 490–440 nm emission was recorded using PhoCal 1.6 b software and calibrated to pHi using the high K+/nigericin technique (Turner et al., 2016) in which cells were exposed to high K+ solutions containing 10 μM nigericin, set to a desired pH, ranging from 6 to 7.5. For analysis of pHi measurements, ΔpHi was determined by calculating the mean pHi over 60 s resulting from treatment. The initial rate of pHi change (ΔpHi/Δt) was determined by performing a linear regression over a period of at least 40 s.

Short-circuit current measurements in human airway epithelial cells

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Cells grown on 6.5 mm inserts were mounted into the EasyMount Ussing Chamber Systems (VCC MC8 Physiologic Instrument, tissue slider P2302T) and bathed in bilateral Cl- free HCO3- KRB (25 mM NaHCO3, 115 mM Nagluconate, 2.5 mM K2SO4, 6.0 mM Ca-gluconate, 1 mM Mg-gluconate, 5 mM D-glucose) continuously gassed and stirred with 5% (v/v) CO2/95% (v/v) O2 at 37 °C or in bilateral NaHEPES buffered solution continuously gassed and stirred with 100% O2 at 37 °C. Monolayers were voltage-clamped to 0 mV and monitored for changes in short-circuit current (ΔIsc) using Ag/AgCl reference electrodes. The transepithelial short-circuit current (Isc) and the Transepithelial electrical resistance (Rte, expressed in Ω cm2) were recorded using Ag–AgCl electrodes in 3 M KCl agar bridges, as previously described (Saint-Criq et al., 2013), and the Acquire & Analyze software (Physiologic Instruments) was used to perform the analysis. Cells were left to equilibrate for a minimum of 10 min before amiloride (10 μM, apical, Sigma-Aldrich #A7410) and S0859 (30 μM, basolateral) were added. Results were normalized to an area of 1 cm2 and expressed as Isc (μAmp.cm–2). The number of replicates was determined using previously obtained short circuit current measurements.

ASL pH measurements

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ASL pH measurements were performed as previously described (Delpiano et al., 2018; Saint-Criq et al., 2019). Briefly, cells grown on 6.5 mm transwells were washed apically with modified Krebs solution for 15 min at 37 °C, 5% CO2. The ASL was stained using 3 μl of a mixture of dextran-coupled pH-sensitive pHrodo Red (0.5 mg/ml, λex: 565 nm, λem: 585 nm; ThermoFisher Scientific, #P10361) and Alexa Fluor 488 (0.5 mg/ml, λex: 495 nm, λem: 519 nm; ThermoFisher Scientific #D-22910) diluted in glucose-free modified Krebs buffer, overnight at 37 °C, 5% CO2. The next day, fluorescence was recorded, every 5 min, using a temperature and CO2-controlled plate reader (TECAN SPARK 10 M) and forskolin (Tocris Biotechne #1099) and S0859 were added basolaterally at indicated times. The ratio of pHrodo to Alexa Fluor 488 was converted to pH using a calibration curve obtained by clamping apical and basolateral pH in situ using highly buffered solutions between 5.5 and 8 (Delpiano et al., 2018). To prevent inter-experiment variability, the standard curve calibration was performed on each independent experiment. Changes in ASL pH (ΔASLpH) were calculated by averaging five time points (average pH over 25 min) before and 2 hrs after the addition of the molecules (Fsk/S0859). The number of replicates was determined using previously obtained ASL pH data (Saint-Criq et al., 2019). Using Cohen’s d, a power analysis showed that the sample size of 9 independent experiments has an 80% power to detect an effect size of 0.35 pH unit, assuming a 5% significance level and a two-sided test (baseline ASL pH = 6.82 ± 0.25).

RNA extraction and PCR analysis

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RNA isolation from cells was performed using PureLink RNA Mini Kit (Ambion, Life technologies, #12183018 A), following the manufacturer’s instructions. Briefly, lysates were mixed with 70% ethanol and loaded onto a silica-membrane column. Columns were washed with different buffers and total RNA was eluted in DNAse and RNAse-free water and stored at –80 °C until use. DNase treatment was performed on 300 ng RNA prior to Reverse Transcription Polymerase Chain Reaction (RT-PCR) using RNAse-free DNAse I (Roche, # 04716728001) at 37 °C for 10 min. Reaction was then stopped by increasing the temperature to 70 °C for 10 min. Complementary DNA (cDNA) was synthesized from total RNA (300 ng) using M-MLV Reverse Transcriptase (Promega, #M1701) as per supplier’s protocol (1 hr at 37 °C followed by 10 min at 70 °C). Expression of SLC4A family members was evaluated by PCR using specific primers (Supplementary file 1), in a total volume of 25 μL containing 2 μL cDNA template, 5 μL 5 X Q5 reaction buffer, 0.5 μL 10 mM dNTP, 1.25 μL 10 μM of each primer, 0.25 μL Q5 high Fidelity Polymerase (New England Biolabs Inc, M0491), 5 μL 5 X Q5 high enhancer (Denaturation, 98 °C, 30 sec; 98 °C 5 sec, 72 °C 30 sec –72 °C 20 sec) x35 cycles; Final Extension, 72 °C, 2 min. PCR products were loaded onto a SYBR Safe DNA stain (Life Technologies, cat. no. S33102)-containing, 2% agarose gels in TBE and electrophoresis was ran at 90 V for 1.5 hr. Amplified products were visualized using LAS-3000 Imaging System (Fuji).

Ex vivo murine airway studies

Animals

The Slc4a4-/- mice was obtained from its laboratory of origin (Gawenis et al., 2007) and bred in the original 129S6/SvEv/Black Swiss background or C57BL/6 J. As observed in Figure 4—figure supplement 1A-B animals maintained in the C57BL/6 J background were severely affected by weight loss and lethality before weaning (day 21 post birth). Therefore experiments were performed in the hybrid animals. The wild type C57BL/6 J mice were from The Jackson Laboratories (USA). Animals were bred and maintained in the Specific Pathogen Free mouse facility of Centro de Estudios Científicos (CECs) with access to food and water ad libitum. 8–12 weeks-old or 16–20 days-old, male or female mice were used. Unless otherwise stated, all procedures were performed after mice were deeply anesthetized via i.p. injection of 120 mg/kg ketamine and 16 mg/kg xylazine followed by exsanguination. All experimental procedures were approved by the Centro de Estudios Científicos (CECs) Institutional Animal Care and Use Committee (#2015–02) and are in accordance with relevant guidelines and regulations.

Ussing chamber experiments

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Tracheae were placed in P2306B of 0.057 cm2 (Figure 3A–K and Figure 4—figure supplement 1A-F) or P2307 of 0.031 cm2 (Figure 4E) tissue holders and placed in Ussing chambers (Physiologic Instruments Inc, San Diego, CA, USA). Tissues were bathed with bicarbonate-buffered solution (pH 7.4) of the following composition (in mM): 120 NaCl, 25 NaHCO3, 3.3 KH2PO4, 0.8 K2HPO4, 1.2 MgCl2, 1.2 CaCl2 or HEPES buffer: 130 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 Na-HEPES (pH adjusted to 7.4 using HCl); supplemented with 10 D-Glucose, gassed with 5% CO2–95% O2 (bicarbonate buffer) or 100% O2 (HEPES buffer) and kept at 37 °C. The transepithelial potential difference referred to the serosal side was measured using a VCC MC2 amplifier (Physiologic Instruments Inc). The short-circuit currents were calculated using the Ohm’s law as previously described (Vega et al., 2020). Briefly electrogenic Na+absorption was inhibited using 10 µM amiloride (Sigma #A7410), cAMP-dependent anion secretion was induced using an IBMX +Forskolin mixture of 100 µM IBMX (Sigma #I5879)+1 µM forskolin (Sigma #F6886), Ca2+-dependent anion secretion was induced by 100 µM UTP (Sigma #U6750). Acetazolamide (100 µM) was used to inhibit bicarbonate production (Sigma #A7011), to block SLC4A4 30 µM S0859 was used (kindly donated by Juergen Puenter, Sanofi-Aventis, France and dissolved in ethanol), and 30 µM CACCinhA01 (Calbiochem) to inhibit CFTR and TMEM16A channels. The ΔIsc values were calculated by subtracting Isc values values before from Isc values after the addition of drugs but UTP induced current was calculated as the area under the curve (A.U.C.) of the first 5 min post UTP addition using the Acquire & Analyze 2.3 v software. Tissues with Rte values below 50 Ωcm2 were discarded as they were not suitable for bona fide electrophysiological determinations (Gianotti et al., 2016).

Airway cells isolation and intracellular pH determinations

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Tracheae were incubated with Pronase 25 µg/ml at 37 °C for 30 min. Then the trachea was placed in a petri dish with DMEM-F12, and the airway epithelium was dissociated by scrapping with tweezers, the cells were collected and spun at 3000 r.p.m. for 5 min at room temperature and the supernatant was removed. The cell pellet was incubated with 500 µl of trypsin 1 X at 37 °C for 5 min and centrifuged at 3000 r.p.m for 5 min at room temperature and the supernatant was removed. The airway epithelial cells were resuspended into 150 µl of DMEM-F12 medium supplemented with 10% fetal bovine serum (FBS) and seeded on poly-L-lysine coated 25 mm glass coverslips in 35 mm Petri dishes. Freshly isolated cells from mouse trachea were loaded with 0.5 µM BCECF-AM (ThermoFisher Scientific #B-1170) for 10 min at 37 °C. After loading, cells were washed and incubated 30 min in imaging solution (see below) to allow probe de-esterification. Cells were mounted into an open chamber and superfused with a bicarbonate buffer imaging solution of the following composition (in mM): 120 NaCl, 25 NaHCO3, 3.3 KH2PO4, 0.8 K2HPO4, 1.2 MgCl2, 1.2 CaCl2 and bubbled with air/5% CO2. For experiments without bicarbonate, the same HEPES buffer as in Ussing chamber experiments was used and bubbled with 100% O2. In the low chloride bicarbonate buffer, 120 mM NaCl was replaced by 115 mM Na-Gluconate and 5 mM NaCl. Experiments were carried out on an Olympus IX70 inverted microscope equipped with a 40 X oil-immersion objective (NA 1.3), a monochromator (Cairn, UK) and a CCD Hamamatsu Orca camera (Hamamatsu, Japan), controlled by Kinetics software. All solutions were superfused at 37 °C using an in-line heating system (Warner instruments). BCECF was excited sequentially at 490 nm and 440 nm for 0.05–0.1 s and emission collected at 535/15 nm. The F490/F440 ratio was computed and transformed to pH units by performing a pH-clamp. Briefly, cells were exposed to 5 µM nigericin and 20 µg/ml gramicidin in a buffer composed of (in mM) 10 HEPES, 129 KCl, 10 NaCl, 1.25 MgCl2, 1 EGTA, 10 glucose, with pH values ranging between 6.8–7.8 and the observed changes in fluorescence were quantified and used to construct a calibration curve.

Airway cell isolation and RT-PCR

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Tracheae were incubated with Pronase 25 µg/ml at 37 °C for 30 minutes. Trachea was placed in DMEM-F12 with 10 mM D-glucose and epithelium was isolated by scrapping with tweezers and further homogenized in 250 µl Trizol (Trizol Reagent) and RNA isolated following the manufacturer´s instructions. The dried pellet of RNA was resuspended with 35 µl nuclease free water and stored at –80 °C. Genomic DNA was removed through DNase treatment. The concentration and integrity of RNA isolation was verified using a NanoDrop spectrophotometer Maestrogen. RNA was reverse transcribed into cDNA using the ImProm-IITM Reverse Transcription System (Promega) following manufacturer’s recommendations. The specific primer pairs used for Slc4a4, Slc4a5, Slc4a7, Slc4a8, Slc4a10, Slc4a4-A, and Slc4a4-B PCR amplification are provided in Supplementary file 2. PCR amplification was performed starting with 3-min template denaturation step at 95 °C, followed by 40 cycles of denaturation at 95 °C for 30 s and combined primer annealing/extension at temperature as appropriate.

Histology and immunofluorescence

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Human tissues were obtained from the Pathological Anatomy Subdepartment of Hospital Base Valdivia (Valdivia, Chile) and corresponded to surgical resections of lung tumors that contained normal parenchyma including epithelium. The studies were approved by the Comité Ético Científico of the Servicio de Salud Los Ríos (CEC-SSV 443–2021). To obtain mice tissues the animals were placed in a 1 litre induction chamber under 1000 ml min−1 flow of air containing 2.5% isoflurane. Then kept under anaesthesia with 2% isoflurane at a constant flow rate of 500 ml min−1 using a mask. Mice were euthanized by exsanguination by severing the inferior vena cava under deep anaesthesia. Mice were perfused with 4% paraformaldehyde (PFA). Trachea and lung were removed and incubated overnight in 4% PFA at 4 °C. Paraffin sections (4 μm) were treated with Trilogy 1 X (Cell Marque cat# 920 P-06), blocked with 2.5% normal goat serum (Vector Laboratories cat# S-1012), and incubated with 1:100 anti-NBCe1 (anti SLC4A4; Alomone cat#ANT-075) 4 °C overnight. Sections were incubated with secondary antibody 1:2000 anti-rabbit Alexa Fluor 488 (Invitrogen cat# A-11008) 2 hr at room temperature. For colocalization 1:100 anti-NBCe1 was incubated with 1:1000 anti-Clara Cell Secretory Protein (CCSP; Merck Millipore cat#07–623) or 1:200 anti- alpha tubulin (Santa Cruz cat#sc-5286) overnight at 4 °C, and incubated with secondary antibody 1:2,000 anti-rabbit Alexa Fluor 568 (Life Technologies cat#A-11011) or 1:2000 anti-mouse Alexa Fluor 568 (Life Technologies cat#A-11004) respectively. Nuclei were stained with 1:2000 propidium iodide (Invitrogen cat#P21493). All immunofluorescence images were captured using a confocal microscope (Olympus FV1000).

Whole-mount trachea Alcian blue cartilage staining

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Tissue was fixed in 95% ethanol overnight followed by 2 hr staining with 0.03% Alcian blue (Sigma cat#A5268) dissolved in 80% ethanol and 20% acetic acid. Samples were cleared in 2% KOH, and pictures taken under a stereomicroscope.

Mucociliary clearance determination

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Speed of polystyrene beads in trachea samples was determined as previously described (Vega et al., 2020). Briefly, the tracheas were isolated and mounted with insert needles onto extra thick blot paper (Bio-Rad) and transferred into a water-saturated chamber at 37 °C. The filter paper was perfused with HCO3- buffered solution of the following composition (in mM): 120 NaCl, 25 NaHCO3, 3.3 KH2PO4, 0.8 K2HPO4, 1.2 MgCl2, 1.2 CaCl2 (gassed with 5% (v/v) CO2/95% (v/v) O2 to maintain solution pH close to 7.4) at a rate of 1 ml min–1 and at 37 °C. Polystyrene black dyed microspheres (diameter 6 µm, 2.6% solid-latex, Polybead, Polyscience Inc) were washed and resuspended in HCO3- or HEPES solution and 4 µl of particle solution with 0.3% latex were added onto the mucosal surface of the trachea. Particle transport was visualized every 5 s for 15 min using a ZEISS SteREO Discovery.V12, with digital camera Motic (Moticam 5.0). Particle speed was calculated with NIH ImageJ software and speed of MCC was expressed in µm/s. To inhibit HCO3- secretion, HCO3- solution was substituted for the HCO3--free solution (HEPES) of the following composition (in mM): 145 NaCl, 1.6 K2HPO4, 0.4 KH2PO4, 1.0 MgCl2, 1.3 CaCl2 throughout the experiments. In these experiments, the HCO3- free solution (HEPES) was gassed with 100% O2 gas to maintain solution pH close to 7.4. Beads tracked on each tissue were averaged and were used as corresponding to one tissue sample.

Statistical analysis

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Experiments in human cells were analysed using GraphPad Prism v9. Statistical analysis was performed taking into account the number n of independent repetitions (done in different days) of the experiments (stated in the Figure legends) using cells at different passage numbers from 3 different donors. Thus n numbers given in the figure legends are considered as biological replicates. Statistical tests used are indicated in the figure legends and p-values are shown in the figures. Experiments using animals were analysed using the Sigmaplot 12 software. Statistical analysis was performed considering as n the number of animals used as source for tissues. These n numbers given on figure legends are considered biological replicates. For pHi experiments isolated cells were from at least 3 different animals per group and statistical analysis was performed using values from individual cells. ANOVA on Ranks was used for comparisons of more than 2 data sets while Rank Sum test for comparison of data with two data sets. For survival analysis Log Rank test was used. p-Value of <0.05 was considered statistically significant. Sample size for experiments in human cells were calculated using Cohen’s d, a power analysis showed that the sample size of five independent experiments has a 90% power to detect a difference, assuming a 5% significance level and a two-sided test. Calculations were based in previously published data for intracellular pH (Turner et al., 2016), ASL pH measurements (Saint-Criq et al., 2019) and unpublished data set for short-circuit currents. Sample size for animal experiments was calculated using previous published data for Ussing chamber experiments (Vega et al., 2020; Gianotti et al., 2016), intracellular pH (Theparambil and Deitmer, 2015) and mucociliary clearance (Vega et al., 2020).

Data availability

All data generated or analysed during this study are included in the manuscript and supporting file.

References

    1. Jia CE
    2. Jiang D
    3. Dai H
    4. Xiao F
    5. Wang C
    (2016)
    Pendrin, an anion exchanger on lung epithelial cells, could be a novel target for lipopolysaccharide-induced acute lung injury mice
    American Journal of Translational Research 8:981–992.
  1. Book
    1. Randell SH
    2. Fulcher ML
    3. O’Neal WK
    4. Olsen JC
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    Primary Epithelial Cell Models for Cystic Fibrosis Research
    Springer International Publishing.

Decision letter

  1. László Csanády
    Reviewing Editor; Semmelweis University, Hungary
  2. Richard W Aldrich
    Senior Editor; The University of Texas at Austin, United States
  3. László Csanády
    Reviewer; Semmelweis University, Hungary
  4. Hugo de Jonge
    Reviewer

Our editorial process produces two outputs: (i) public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.

Decision letter after peer review:

Thank you for submitting your article "Inhibition of the sodium-dependent HCO3 -transporter SLC4A4, produces a cystic fibrosis-like airway disease phenotype." for consideration by eLife. Your article has been reviewed by 3 peer reviewers, including László Csanády as Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Richard Aldrich as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Hugo de Jonge (Reviewer #2).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

1. SLC4A4, A5 and A8 are the major NCBTs expressed in the primary hAECs (Figure S1A). The arguments used to exclude A5 and A8 as candidate HCO3- importers (l. 298-301) are weak because (i) the fact that transepithelial HCO3- transport generates a Isc does not prove that the importer is electrogenic (cf. Cl- secretory currents mediated through the electroneutral NKCC1 importer), and (ii) the localization of SLC4A5 in the apical membrane of renal epithelial cells does not rule out a basolateral localization in airway cells (cf. the opposite polarization of AE2 in intestinal vs. bile duct cells and of CA12 in salivary duct vs. airway surface cells). Does the SLC4A4 inhibitor S0859, at 30 μm concentration, also inhibit A5 or A8? Please comment. Furthermore, the authors report that both SLC4A7 and SLC4A10 had very low expression levels, although in Figure S1A it looks as if the expression of SLC4A7 could be quite reasonable. How did the authors determine relative mRNA abundance and, along similar lines, were the amplified bands digested to confirm their identity? Finally, are other candidate HCO3- transporters or channels, e.g. Bestrophins, expressed too in primary hAECs?

2. Figure 1A-D: The size of the S0859-inhibitable Isc (representing a HCO3- secretory current) in the hAECs is very small (~1 uAmp/cm2) in comparison with anion current measurements in similar cell models reported previously (e.g. refs. 14, 27: ~20 uAmp/cm2). This is remarkable because the conditions used (Cl- free buffer) is optimal for de-inhibition of NBCe1-B activity that is known to act as a Cli sensor through its 2 GXXXP motifs (Shcheynikov et al. 2015 PNAS 112: E329-37). To judge better about the quality of the hAEC monolayers in culture, one would like to know how the inclusion of Cl-, or CFTR activation by forskolin in the perfusion medium (affecting the ASL pH; Figure 2) affects Isc measurements in the Ussing chamber, and to what extent S0859 inhibits forskolin-stimulated HCO3- currents.

3. Figure 2, G-J: In the hAEC monolayers SLC4A4 seems to be co-expressed with acetylated tubulin in ciliated cells. However it is unclear how many other cell types (CC10-positive Club cells; ionocytes; goblet cells) are retrieved in these human airway cell cultures. In both mouse bronchi and bronchioles (Suppl. Figure 4C) and in differentiated hAECs in the Welsh lab (ref. 15) CFTR (and pendrin) are expressed mainly in CC10+-secretory (Club) cells, not in ciliated cells. This raises the question whether SLC4A4 is also expressed in human Club cells and whether Club cells rather than ciliated cells are the major sources of HCO3- secreted into the ASL.

4. Figure 3 A-C: Can the authors explain why the IBMX/forskolin/cAMP-induced anion current, in contrast to the UTP/ca2+-induced current, is not reduced in bicarbonate-free buffer, i.e. does not seem to have a HCO3- component? In mouse trachea, most if not all Fsk/cAMP-induced anion secretory current is CaCC-mediated, so there is a priori no reason why forskolin-induced currents would not be reduced (at least in part) in a HCO3- free buffer.

5. Figure S3D-F: Assuming that the CaCC inhibitor AO1 completely blocks CaCCs and the IBMX/Fsk-induced Isc, how do the authors explain that subsequent addition of S0859 further inhibits a "basal" HCO3- secretory current? Does HCO3- exit the cell at the apical side through tonically active CFTR, or through an electrogenic HCO3-/Cl- exchanger (but pendrin is electroneutral)?

6. Figure 3, D-E, Figure S3A and D, and l. 350: The cause of the transient negative change in Isc elicited by S0859 remains to be identified. Could it be that the compound has transient off-target effects on the intracellular ca2+ level, or activates basolateral K+ channels? It is remarkable that similar deflections are not seen in human AECs (Figure 1C).

7. How close do the hAECs used in this study recapitulate the airways in situ? For example, are CA12 (cf. ref. 11) or ATP12A (cf. ref. 7) highly expressed in these cultures? This could either be addressed using Q-PCR of markers, e.g. CA12 and ATP12A, or by immunostaining of SLC4A4 in CC10+ cells.

8. The systemic Slc4a4 null-mouse model used in this study is known to suffer from many abnormalities, including severe metabolic acidosis due to pRTA (l. 471-480). Whether the reduced level of plasma HCO3- contributes to the lack of HCO3- secretion and reduced ASL pH in the trachea of the Slc4a4-/- mouse (l. 475) depends on the affinity of the NBCe1 transporter for HCO3- ions. Therefore it is of interest to learn whether the transporter is already saturated at 5 mM HCO3-, or needs higher serosal HCO3- levels, e.g. 24 mM. This could be addressed experimentally or at least discussed.

9. L. 496: I agree that an airway-specific Slc4a4 null mouse model created by Cre-Lox technology would be of further help in studying the pathogenesis of muco-obstructive diseases of the lungs. In particular a comparison of Club cell-specific vs. ciliated cell-specific Slc4a4-/- mouse models would be useful to study the functional importance of SLC4A4 in each cell type in vivo.

10. The authors show changes in ASL pH in hAEC cultures. Could the authors include in their Methods a statement about the time point at which the pH was considered to have settled, i.e. at what time were the Figure 2B data points collected following the addition of S0859 or Fsk? Could the authors also comment on the difference in timescale between the effects on intracellular acidification (Figure 1) vs the effect on ASL pH which seems to take ~2hrs (Figure 2)?

11. It looks as if some effects are not fully reversible (Figure S3G)? Could the authors comment on this?

12. In relation to the central thesis that pH is central in the development of the CF mucus phenotype, is there any evidence of changes to mucus rheology in the hAEC cultures accompanying the changes in pH?

13. The authors pursue an experiment to determine whether there is "an active NCBT under resting conditions". But surely Isc measurements are not really made under resting conditions as the ASL is essentially flooded and the ionic composition on the apical side is not likely to match that of a normal ASL. This will affect ion driving forces etc. Could the authors consider this point?

Reviewer #1 (Recommendations for the authors):

1. Figure 1A vs. C: Why did the authors change two parameters at the same time, by switching from Cl- free HCO3- containing (in A) to Cl- containing HCO3- free solution (in C)? Wouldn't a Cl- free HEPES-buffered solution be a better control for the experiment in A? Also, please indicate the timepoint of basolateral drug addition in panels A, C.

2. Figure 2C: In the cells pretreated with S0859 Fsk exposure seems to induce a transient small acidification. When compared to that acidified value, the ensuing pH recovery seems comparable to that observed for cells not pretreated with S0859. Can the authors comment on the likely mechanism of this phenomenon?

3. Figure 2B, D, F: Please define what the symbols represent. Is it pH at a given (which?) timepoint following drug addition? Or mean pH observed over a given (which?) time interval following drug addition?

4. Figure 2B, D, F: The plotted significance values are confusing. If the error bars on the black and blue dot represent S.E.M., than the difference is unlikely to be significant in either panel. Was the significance calculated for the δ-pH values? If so, it would be helpful to also provide mean+/-S.E.M. δ-pH, and indicate the significance value there.

5. Figure 3C: mean+/-S.E.M. bars are hidden behind the individual data points.

6. Figure 3L-M: in panel L please show the data also for the third bar in panel M.

7. Suppl. Figure 3C, F: It is unclear what the bars represent. E.g., in panel C the two bars seem identical (negative), although in panels A-B S0859 induces a positive current change when added after Fsk (i.e., δ-Isc should be positive for S0859). The same applies for the 2nd and 3rd bars in panel F.

8. Suppl. Figure 3G: Several clarifications are needed here:

(i) The legend says "UTP-induced intracellular acidification" but the trace seems to show S0859-induced acidification.

(ii) The legend says "in bicarbonate buffer or HEPES", but in the trace it is not indicated where the buffer change has occurred.

(iii) Please calibrate the ordinate into pH units for a better comparison with panel H.

Reviewer #2 (Recommendations for the authors):

1. SLC4A4, A5 and A8 are the major NCBTs expressed in the primary hAECs (Figure S1A). In my view the arguments used to exclude A5 and A8 as candidate HCO3- importers (l. 298-301) are weak because (i) the fact that transepithelial HCO3- transport generates a Isc does not prove that the importer is electrogenic (cf. Cl- secretory currents mediated through the electroneutral NKCC1 importer), and (ii) the localization of SLC4A5 in the apical membrane of renal epithelial cells does not rule out a basolateral localization in airway cells (cf. the opposite polarization of AE2 in intestinal vs. bile duct cells and of CA12 in salivary duct vs. airway surface cells). Does the SLC4A4 inhibitor S0859, at 30 μm concentration, also inhibit A5 or A8? Are other candidate HCO3- transporters or channels , e.g. Bestrophins, expressed too in primary hAECs?

2. Figure 1A-D: The size of the S0859-inhibitable Isc (representing a HCO3- secretory current) in the hAECs is very small (~1 uAmp/cm2) in comparison with anion current measurements in similar cell models reported previously (e.g. refs. 14, 27: ~20 uAmp/cm2). This is remarkable because the conditions used (Cl- free buffer) is optimal for de-inhibition of NBCe1-B activity that is known to act as a Cli sensor through its 2 GXXXP motifs (Shcheynikov et al. 2015 PNAS 112: E329-37). To judge better about the quality of the hAEC monolayers in culture, one would like to know how the inclusion of Cl-, or CFTR activation by forskolin (affecting the ASL pH; Figure 2) in the perfusion medium affects Isc measurements in the Ussing chamber, and to what extent S0859 inhibits forskolin-stimulated HCO3- currents.

3. Figure 2, G-J: In the hAEC monolayers SLC4A4 seems to be co-expressed with acetylated tubulin in ciliated cells. However it is unclear how many other cell types (CC10-positive Club cells; ionocytes; goblet cells) are retrieved in these human airway cell cultures. In both mouse bronchi and bronchioles (Suppl. Figure 4C) and in differentiated hAECs in the Welsh lab (ref. 15) CFTR (and pendrin) are expressed mainly in CC10+-secretory (Club) cells, not in ciliated cells. This raises the question whether SLC4A4 is also expressed in human Club cells and whether Club cells rather than ciliated cells are the major sources of HCO3- secreted into the ASL.

4. Figure 3 A-C: Can the authors explain why the IBMX/forskolin/cAMP-induced anion current, in contrast to the UTP/ca2+-induced current, is not reduced in bicarbonate-free buffer, i.e. does not seem to have a HCO3- component? In mouse trachea, most if not all Fsk/cAMP-induced anion secretory current is CaCC-mediated, so there is a priori no reason why forskolin-induced currents would not be reduced (at least in part) in a HCO3- free buffer.

5. Figure S3D-F: Assuming that the CaCC inhibitor AO1 completely blocks CaCCs and the IBMX/Fsk-induced Isc, how do the authors explain that subsequent addition of S0859 further inhibits a "basal" HCO3- secretory current? Does HCO3- exits the cell at the apical side through tonically active CFTR, or through an electrogenic HCO3-/Cl- exchanger (but pendrin is electroneutral)?

6. Figure 3, D-E, Figure S3A and D, and l. 350: The cause of the transient negative change in Isc elicited by S0859 remains to be identified. Could it be that the compound has transient off-target effects on the intracellular ca2+ level, or activates basolateral K+ channels? It is remarkable that similar deflections are not seen in human AECs (Figure 1C).

7. How close do the hAECs used in this study recapitulate the airways in situ? For example, are CA12 (cf. ref. 11) or ATP12A (cf. ref. 7) highly expressed in these cultures?

8. The systemic Slc4a4 null-mouse model used in this study is known to suffer from many abnormalities, including severe metabolic acidosis due to pRTA (l. 471-480). Whether the reduced level of plasma HCO3- contributes to the lack of HCO3- secretion and reduced ASL pH in the trachea of the Slc4a4-/- mouse (l. 475) depends on the affinity of the NBCe1 transporter for HCO3- ions. Therefore it is of interest to learn whether the transporter is already saturated at 5 mM HCO3-, or needs higher serosal HCO3- levels, e.g. 24 mM.

9. L. 496: I agree that an airway-specific Slc4a4 null mouse model created by Cre-Lox technology would be of further help in studying the pathogenesis of muco-obstructive diseases of the lungs. In particular a comparison of Club cell-specific vs. ciliated cell-specific Slc4a4-/- mouse models would be useful to study the functional importance of SLC4A4 in each cell type in vivo.

In my view the paper could be strengthened further by:

1. In general: addressing the points of discussion raised in the public review and incorporating some of the answers in the paper.

2. Providing additional information about the cell composition, Cl- vs. HCO3- currents, and possible roles of Slc4A5 and A8 (both highly expressed) in the hAECs.

3. If available, grow primary hAECs under the conditions described in ref. 15 (enriched in CC10+ Club cells) and determine whether NBCe1-B is expressed in the ciliated cells, in the Club cells or in both.

4. Find out whether Slc4A4 becomes already saturated at 5 mM HCO3-, or needs higher serum HCO3- levels for optimal function.

Reviewer #3 (Recommendations for the authors):

1. The authors show some nice amplification of SLC4A bands and report that both SLC4A7 and SLC4A10 had very low expression levels, referring the reader to Figure S1A. However, it looks as if the expression of SLC4A7 could be quite reasonable in this figure. How did the authors determine relative mRNA abundance and, along similar lines, were the amplified bands digested to confirm their identity?

2. The authors provide evidence supporting the expression of several members of the SLC4A family of transporters in human airway epithelial cells. Although S0859 is a blocker of SLC4A4, is it selective for SLC4A4 or is it possible other members of the SLC4A family might be blocked by this drug and contributing to some of the observed effects e.g. ASL pH changes? Can the authors comment on this?

3. The authors show changes in ASL pH in hAEC cultures. Could the authors include in their Methods a statement about the time point at which the pH was considered to have settled, i.e. at what time were the Figure 2B data points collected following the addition of S0859 or Fsk? Could the authors also comment on the difference in timescale between the effects on intracellular acidification (Figure 1) vs the effect on ASL pH which seems to take ~2hrs (Figure 2)?

4. It looks as if some effects are not fully reversible (Figure S3G)? Could the authors comment on this?

5. Direct measurements of ASL volume and pH in the knockout mice would clearly strengthen part of the central thesis, although these measurements are admittedly difficult to perform. However, on a related topic, is there any evidence of changes to mucus rheology in the hAEC cultures accompanying the changes in pH?

6. The authors pursue an experiment to determine whether there is "an active NCBT under resting conditions". But surely Isc measurements are not really made under resting conditions as the ASL is essentially flooded and the ionic composition on the apical side is not likely to match that of a normal ASL. This will affect ion driving forces etc. Could the authors consider this point?

https://doi.org/10.7554/eLife.75871.sa1

Author response

Essential revisions:

1. SLC4A4, A5 and A8 are the major NCBTs expressed in the primary hAECs (Figure S1A). The arguments used to exclude A5 and A8 as candidate HCO3- importers (l. 298-301) are weak because (i) the fact that transepithelial HCO3- transport generates a Isc does not prove that the importer is electrogenic (cf. Cl- secretory currents mediated through the electroneutral NKCC1 importer), and (ii) the localization of SLC4A5 in the apical membrane of renal epithelial cells does not rule out a basolateral localization in airway cells (cf. the opposite polarization of AE2 in intestinal vs. bile duct cells and of CA12 in salivary duct vs. airway surface cells). Does the SLC4A4 inhibitor S0859, at 30 μm concentration, also inhibit A5 or A8? Please comment. Furthermore, the authors report that both SLC4A7 and SLC4A10 had very low expression levels, although in Figure S1A it looks as if the expression of SLC4A7 could be quite reasonable. How did the authors determine relative mRNA abundance and, along similar lines, were the amplified bands digested to confirm their identity? Finally, are other candidate HCO3- transporters or channels, e.g. Bestrophins, expressed too in primary hAECs?

We thank the reviewers for their important comment on the potential of other SLC4A family members to be involved in intracellular as ASL pH regulation in airway epithelial cells. After a careful literature review we could not find any decisive evidence whether S0859 specifically inhibits SLC4A4 and not the other SLC4A bicarbonate co transporters (Ch’en et al. Br J Pharmacol. 2008; Schwab et al. J Physiol. 2005). Moreover, although we did not perform qPCR experiments to measure the levels of mRNA of each SLC4A member, RNA-seq data from another study using epithelial cells from the same donors, grown in the same conditions, revealed that SLC4A4 is the most expressed member, closely followed by SLC4A7, SLC4A8 and SLC4A5 (see Figure 1—figure supplement1). No counts were found for SLCA10. Data from this project are reported in the following publication (Saint-Criq et al. Cells. 2020 doi: 10.3390/cells9092137. PMID: 32967385) and the data are available are accessible through Gene Expression Omnibus (GEO) series accession number GSE154905.

We also report the levels of expression of Bestrophins 1, 3 and 4 in primary hAECs. Bestrophins mRNA levels were 1 to 2 log lower than SLC4A4, A7 and A8. Taken together, we believe there is not strong enough evidence that allows us to rule out other members of the SLC4A family, especially SLC4A7 and SLC4A8. Thus, in the manuscript we have added these data as Figure 1—figure supplement1G, edited the main text, and discussed these results further in the Discussion section (lines 228-231).

2. Figure 1A-D: The size of the S0859-inhibitable Isc (representing a HCO3- secretory current) in the hAECs is very small (~1 uAmp/cm2) in comparison with anion current measurements in similar cell models reported previously (e.g. refs. 14, 27: ~20 uAmp/cm2). This is remarkable because the conditions used (Cl- free buffer) is optimal for de-inhibition of NBCe1-B activity that is known to act as a Cli sensor through its 2 GXXXP motifs (Shcheynikov et al. 2015 PNAS 112: E329-37). To judge better about the quality of the hAEC monolayers in culture, one would like to know how the inclusion of Cl-, or CFTR activation by forskolin in the perfusion medium (affecting the ASL pH; Figure 2) affects Isc measurements in the Ussing chamber, and to what extent S0859 inhibits forskolin-stimulated HCO3- currents.

We thank the reviewer for their comment. In Author response image 1 we provide data representing CFTR activation in the presence of HCO3- (HCO3 KRB) and in the absence of Cl- (0Cl-). As shown in Author response image 1, CFTR-dependent currents were significantly reduced (> 90 %) in the absence of Cl-, consistent with previous reports that HCO3- has a much lower permeability (conductance) through CFTR compared to Cl- (Gray et al., Am. J. Physiol.1990; Linsdell et al., JCF, 2009). In addition, the size of the FSK-stimulated Isc in the absence of Cl- is actually very similar to that found by Gorrieri (ref 33) using similar conditions (~2 µAmp/cm2- see Figure 4C, no IL4), and NOT 20uAmp/cm2 which was the value with both Cl and HCO3.

Author response image 1
Comparison of HCO3- KRB and Cl- free solutions on Isc responses of primary hAECs to amiloride, forskolin, potentiator P5 and CFTRInh172.

Panel on the left shows a representative trace. Panels on the right show either baseline Isc (top left) or changes in Isc (ΔIsc) after specific inhibitors and agonists. Each symbol represents an independent experiment, using cells from two donors. Bars are means ± SD.

We have also included in the revised manuscript, the effect of S0859 on Fsk-induced HCO3- secretion in Ussing chamber experiments (Figure 1, panels A and C). In this setting, S0859 did not affect the Fsk-induced change in Isc. This result suggests that there may be other mechanisms that the epithelium uses to accumulate bicarbonate intracellularly in order to maintain Fsk-induced bicarbonate secretion. There are a number of possibilities, including, other NBCs that may not be inhibited by S0859 (see point 1 above), as well as the generation of HCO3- via intracellular carbonic anhydrase (CA), when the NBC is inhibited. We did test the effect of inhibiting CA (using acetazolamide), on unstimulated and Fsk-stimulated HCO3- secretion in Ussing chamber experiments under Cl- free conditions, but did not observe any effect of ACTZ – see Author response image 2. However, we believe that further investigation into this aspect of the work is beyond the scope of this current manuscript, particularly because the 0Cl- condition itself is nonphysiological, and we do not know the full effect of bathing the epithelia in a Cl- free buffer on (i) ion gradients and (ii) epithelial cell homeostasis in general. This is why we studied the effect of NBC inhibition in a more physiologically relevant condition, i.e. under thin film condition using normal physiological solutions (ASL pH measurements) and a murine model. Overall, we believe our results from using both fully differentiated primary human airway epithelial cells and mouse models, strongly support a pivotal role of SLC4A4 in pH airway homeostasis

Author response image 2
Effect of acetazolamide (ACTZ) on unstimulated and Fsk-stimulated HCO3- secretion in Ussing chamber experiments in Cl- free buffer.

Upper panels show the kinetics of the effect of ACTZ. Bottom panels are the DMSO, ACTZ and Fsk-induced changes.

3. Figure 2, G-J: In the hAEC monolayers SLC4A4 seems to be co-expressed with acetylated tubulin in ciliated cells. However it is unclear how many other cell types (CC10-positive Club cells; ionocytes; goblet cells) are retrieved in these human airway cell cultures. In both mouse bronchi and bronchioles (Suppl. Figure 4C) and in differentiated hAECs in the Welsh lab (ref. 15) CFTR (and pendrin) are expressed mainly in CC10+-secretory (Club) cells, not in ciliated cells. This raises the question whether SLC4A4 is also expressed in human Club cells and whether Club cells rather than ciliated cells are the major sources of HCO3- secreted into the ASL.

The samples in Figure 2-G-J correspond to human lung biopsies and not hAEC monolayers. The data from the Welsh paper (15) comes from hAECs incubated with inflammatory cytokines and not naïve cells. It is also important to mention that in the Welsh paper there was no increase in expression of the SLC4A4 as seen with Pendrin after the cytokine incubation of hAECs (Figure 5B; Welsh paper), suggesting that bicarbonate secretion in non-ciliated cells might depend on a different basolateral bicarbonate carrier. Welsh data is also different to the data from the Hanrahan lab (Ref 14) and Boucher lab (Ref 41) that show CFTR and Pendrin to be expressed in naïve ciliated cells, the same cells where we observe SLC4A4. The last two mentioned papers supports our immunolocalization results, that there is: an important role for ciliated cells in bicarbonate secretion and is consistent with our observations in ASL pH (Figure 2). Finally, we believe that it is very valuable to share this staining of SLC4A4, as is the only staining of SLC4A4 using native tissue known to the date. We added the following at lane 252-255:

“Even though, CFTR and Pendrin expression increases in secretory cells after cytokine stimulation, SLC4A4 remained unaltered, suggesting that other basolateral HCO3- transporter support HCO3- secretion in human secretory cells during the inflammatory response(15)”.

4. Figure 3 A-C: Can the authors explain why the IBMX/forskolin/cAMP-induced anion current, in contrast to the UTP/ca2+-induced current, is not reduced in bicarbonate-free buffer, i.e. does not seem to have a HCO3- component? In mouse trachea, most if not all Fsk/cAMP-induced anion secretory current is CaCC-mediated, so there is a priori no reason why forskolin-induced currents would not be reduced (at least in part) in a HCO3- free buffer.

Similar to our results, previous work from the laboratory of Marcus Mall (REF 24;Anagnostopolou et al. 2012 JCI 122(10): 3629-3634; Sup Figure 2) showed that the cAMP-induced current is similar when the experiment is performed in HEPES vs HCO3- buffer in mouse tracheas. To our knowledge Shah et al.,2016 (Science. 2016 Jan 29; 351(6272): 503–507) is the only paper where bicarbonate secretion in mouse airway cell cultures has been shown. The size of the current recorded after cAMP stimulation is very small (around 4 µA cm-2) and the recordings were performed in bath solution with no Cl- to isolate the bicarbonate current. It might be possible that due to the small magnitude of the bicarbonate current the difference is not noticeable when using buffers containing Cl- as in our case and in the mentioned paper of M. Mall’s group. We have added this reference in the description of Results Lane:144-145

“but no significant effect on the cAMP-induced anion secretion, or the amiloride-sensitive sodium absorption (Figure 3C) was detected as previously shown(24).”

5. Figure S3D-F: Assuming that the CaCC inhibitor AO1 completely blocks CaCCs and the IBMX/Fsk-induced Isc, how do the authors explain that subsequent addition of S0859 further inhibits a "basal" HCO3- secretory current? Does HCO3- exit the cell at the apical side through tonically active CFTR, or through an electrogenic HCO3-/Cl- exchanger (but pendrin is electroneutral)?

Previous experiments performed in tracheas of mice showed that the genetic silencing of Tmem16a doesn’t affect the cAMP-induced anion seretion but reduces by nearly 50% the Ca+2induced anionic secretion (Rock et al., J Biol Chem. 2009 May 29; 284(22): 14875–14880; Ousingsawat et al., J Biol Chem. 2009 Oct 16;284(42):28698-703). Our own exploration of anion secretion in mouse airways and hAECs demonstrated that CaCCinhA01 inhibits both TMEM16A and CFTR (Ref 68), suggesting that CaCCinhA01 affects cAMP-induced secretion. Our interpretation of those and our present results is that in mouse trachea there is an unidentified anion channel that participates in anion secretion, but such statement (and aim) is beyond the scope of the present work and would require additional experiments, including the breeding a double null Cftr/Tmem16a mouse, to test the hypothesis.

6. Figure 3, D-E, Figure S3A and D, and l. 350: The cause of the transient negative change in Isc elicited by S0859 remains to be identified. Could it be that the compound has transient off-target effects on the intracellular ca2+ level, or activates basolateral K+ channels? It is remarkable that similar deflections are not seen in human AECs (Figure 1C).

Other authors using hAEC have also observed this transient change in Isc (REF 25; Figure 5 A,B) that we cited in the text lane 163. Is possible to observe such negative deflection in the hAECs monolayer used by us in Figure 1A. As the reviewer suggests S0859 has some off targets so, we have added this information and possibility at Lane 161-164:

“Of note, the addition of S0859 to the tracheas induced a fast and transient negative change in Isc as observed in Figure 3D and E and Figure 3-figure 3 supplement 2A and D, that has been also observed in human cells (25), and that might be due to off-targets of the blocker like other SLC4A or SLC16A transporters as previously described (26-28).”

7. How close do the hAECs used in this study recapitulate the airways in situ? For example, are CA12 (cf. ref. 11) or ATP12A (cf. ref. 7) highly expressed in these cultures? This could either be addressed using Q-PCR of markers, e.g. CA12 and ATP12A, or by immunostaining of SLC4A4 in CC10+ cells.

This is a very important question. To fully answer this we would have to compare levels of expression of multiple airway epithelial cell markers between our cultures and native human lung tissue. At this stage, we are not able to obtain freshly isolated cells from non-CF donors to undertake this comparison. However, as stated previously, we have RNAseq data from a different study, and Author response image 3 shows the normalized counts for some airway epithelial ion channels and transporters. As shown in the figure, and published before, ATP12A is highly expressed in our primary cells (REF 22), whereas expression of CA12 is ~ 2-log units lower, and similar to CFTR and β ENaC. Recent work from Kim et al.,2021 (REF 11), compared relative CA12 mRNA expression between freshly isolated cells from CF bronchus to cultured CF and non-CF ALI grown bronchial cells. They showed that CA12 levels were ~ 2-fold higher in freshly isolated CF cells compared to CF cultured cells, but similar to non-CF cultured cells. However, the expression levels were very donor-dependent in cultured cells, but this issue was not investigated in freshly isolated cells. Overall, this study showed that cultured ALI grown cells were a good model of the airways in situ. However, it is indeed very important to optimize growth conditions as these can affect mRNA and protein levels as well as the function of channels, transporters and enzymes involved in ASL pH homeostasis, as we have previously described (REF 17).

Author response image 3
Normalised counts of ion channels and transporters expressed in primary human airway epithelial cells.

Each symbol represents one donor. Bars are means ± SD.

8. The systemic Slc4a4 null-mouse model used in this study is known to suffer from many abnormalities, including severe metabolic acidosis due to pRTA (l. 471-480). Whether the reduced level of plasma HCO3- contributes to the lack of HCO3- secretion and reduced ASL pH in the trachea of the Slc4a4-/- mouse (l. 475) depends on the affinity of the NBCe1 transporter for HCO3- ions. Therefore it is of interest to learn whether the transporter is already saturated at 5 mM HCO3-, or needs higher serosal HCO3- levels, e.g. 24 mM. This could be addressed experimentally or at least discussed.

We thank the reviewer for this comment. Effectively, our own experiments (REF 56) demonstrate that NBCe1 is a low affinity bicarbonate transporter that is saturated at HCO3- 3 mM, a concentration significantly lower than that observed in the systemic Slc4a4 null-mouse (close to 5 mM; REF 54). Thus the reduction in plasma bicarbonate as observed in pRTA (16-20 mM), or in the KO mouse, largely exceeds the concentration at which the SLC4A4 transporter is saturated (Palmer et al., (2021) Adv Ther 38(2):949-968). To include this observation we have rephrased the Discussion including references at Lane 297-299 “Even though, decreased plasma HCO3- is observed in the Slc4a4-/- mice (5.3 ± 0.5 mM) (54) is unlikely that reduced HCO3- availability influenced mucus accumulation in the animals, as we have previously determined that the transporter is fully saturated at 3 mM HCO3- (56).”

9. L. 496: I agree that an airway-specific Slc4a4 null mouse model created by Cre-Lox technology would be of further help in studying the pathogenesis of muco-obstructive diseases of the lungs. In particular a comparison of Club cell-specific vs. ciliated cell-specific Slc4a4-/- mouse models would be useful to study the functional importance of SLC4A4 in each cell type in vivo.

We are currently breeding the Club cell specific null mouse but the results will only be available in a year or so.

10. The authors show changes in ASL pH in hAEC cultures. Could the authors include in their Methods a statement about the time point at which the pH was considered to have settled, i.e. at what time were the Figure 2B data points collected following the addition of S0859 or Fsk? Could the authors also comment on the difference in timescale between the effects on intracellular acidification (Figure 1) vs the effect on ASL pH which seems to take ~2hrs (Figure 2)?

We thank the reviewer for their comments. We have now added the following statement to the Methods section: Lane: 386-391;

“Changes in ASL pH (ΔASLpH) were calculated by averaging five time points (average pH over 25 min) before and 2hrs after the addition of the chemicals (Fsk/S0859).”

However, we would like to point out that the time taken to reach steady-state was actually less than 2hrs after the addition of Fsk. For comparison purposes, a better representation of the time taken to reach steady-state is to calculate the t1/2 and we have presented these values in Figure 2. The t1/2 for the effect of S0859 on resting and Fsk-stimulated ASL pH was 46 mins and 14 mins, respectively, whereas the t1/2 for the effect of Fsk on resting and S0859inhibited ASL pH was 25 mins and 31 mins, respectively.

With regard to the different time scales between the pHi and ASL pH experiments, we would like to point out that making a ‘direct’ comparison is complicated for a number of reasons. (1) The experimental technique and conditions used to assess pH changes differed between the two measurements. For ASL pH, the cell cultures were maintained at the air-liquid interface with differentiation medium only on the basolateral side of the cultures, which was not perfused. Small aliquots of stock solutions of chemicals were manually added to the basolateral solution. For the pHi experiments, the cell cultures were submerged in buffer and continuously perfused apically and basolaterally. Exchange times for solution changes in these pHi expts were ~ 1 min. (2) For the pHi experiments, intracellular acidification was induced by increasing CO2, whereas for the ASL pH expts, changes in ASL pH were induced by adding agonists/inhibitors to the nonperfused basolateral compartment. In separate (new) experiments, when CO2 levels were acutely increased from 0.1 % to 5.0 %, ASL acidified with a t1/2 of 6.7 mins (n=3 – see Author response image 5), considerably faster than the agonist-induced changes. (3) For the ASL pH experiments, although the apical surface was washed 24hr before experiments started, these epithelial cells produce mucus, which accumulates in the ASL over the 24 hr period, and very likely increases the buffering capacity of the luminal surface, thereby slowing pH changes. Indeed, Kim et al., (JEM, 2021) recently showed that adding purified mucin to washed ALI cultures significantly slowed ASL pH changes induced by CO2 removal by ~ 80%. (4) The cultured cells also express multiple carbonic anhydrases (Author response image 5 – Results from the RNA-seq study), and other transporters that will also impact the kinetics of responses. We are not aware of any reports which have directly compared the activity of cytosolic carbonic anhydrases versus extracellular CAs, but this is also an important factor to consider, as CA activity has pronounced effects on pH kinetics (Kim et al., JEM. 2021; REF 11). Overall, both technically, and experimentally ASL pH changes would be expected to take longer to elicit a response.

Author response image 4
A. Effect of increasing CO2 concentration on ASL pH (black line) of hAECs (n=3, mean ± SD).

Exponential decay nonlinear regression analysis (red dashed line) and simple linear analysis (blue line) were performed on the ASL pH values from the change in CO2 concentration. B. Average rate of CO2induced acidification of 3 independent experiments measured as the slope of the linear regression analysis.

Author response image 5
Normalised counts of carbonic anhydrase (CA) family members expressed in primary human airway epithelial cells.

Each symbol represents one donor. Bars are means ± SD.

11. It looks as if some effects are not fully reversible (Figure S3G)? Could the authors comment on this?

This might be because the binding of the inhibitor to the SLC4A4 transporter cannot be fully washed off. However we found reversibility to be variable between experiments. We have changed the figure for a different trace where it is possible to observe that the effect of S0859 is more reversible than in the original trace. Figure 3—figure supplement 2G.

12. In relation to the central thesis that pH is central in the development of the CF mucus phenotype, is there any evidence of changes to mucus rheology in the hAEC cultures accompanying the changes in pH?

We have not measured mucus rheology in hAECs after treatment with S0859. However, Tang et al. 2016 (JCI 126(3); 879-891; REF 49). They observed increased viscosity of the ASL (including mucins) obtained from cell cultures of human AECs and pig AECs affected by CF (FIGURE 2). The increased viscosity parallels with a lower pH compared with cultured cells isolated from non-CF pigs (FIGURE 4). We have added this evidence and corresponding reference in the Discussion at Lane 283-284

“For example, hAEC from CF patients produce acidic ASL and mucus that is more viscous than in cells from non-CF donors (49).”

13. The authors pursue an experiment to determine whether there is "an active NCBT under resting conditions". But surely Isc measurements are not really made under resting conditions as the ASL is essentially flooded and the ionic composition on the apical side is not likely to match that of a normal ASL. This will affect ion driving forces etc. Could the authors consider this point?

We agree with the reviewer that these conditions did not represent resting conditions. We have amended the text and replaced ‘resting’ by ‘unstimulated’

Reviewer #1 (Recommendations for the authors):

1. Figure 1A vs. C: Why did the authors change two parameters at the same time, by switching from Cl- free HCO3- containing (in A) to Cl- containing HCO3- free solution (in C)? Wouldn't a Cl- free HEPES-buffered solution be a better control for the experiment in A? Also, please indicate the timepoint of basolateral drug addition in panels A, C.

These conditions were selected in order to specifically isolate Cl- and HCO3- currents. Although a Cl- free HEPES buffered solution would have indeed been a complementary control condition, we wanted to specifically evaluate the impact of S0859 on the HCO3- current alone and Cl- current, independently of the other anion. Dotted lines have been added to Figure 1A and 1C to indicate the drug addition.

2. Figure 2C: In the cells pretreated with S0859 Fsk exposure seems to induce a transient small acidification. When compared to that acidified value, the ensuing pH recovery seems comparable to that observed for cells not pretreated with S0859. Can the authors comment on the likely mechanism of this phenomenon?

We believe the transient acidification seen in the S0859 treated cell was an artefact, as this did not occur in all experiments, as seen in Author response image 6. This figure shows individual ASL pH recordings in S0859-treated hAECs obtained from 9 independent experiments. We can see that only two experiments (panels D and E) showed a transient acidification following forskolin addition to the basolateral medium. Note that the peaks observed at the time of addition of forskolin are due to the drop in CO2 (from 5% to air) when the plates were removed from the plate-reader in order to treat the cells.

If it isn’t an artefact, this acidification could be due to concurrent Fsk-stimulated H+ secretion (as shown by Shah et al., (2016) in pig and human airway epithelial cells in the absence of HCO3/CO2). In DMSO treated cells, this would be masked by HCO3- secretion (driven by accumulated intracellular HCO3-). In S0859-treated cells, cytoplasmic HCO3- accumulation is decreased, and thus H+ secretion into the ASL would be transiently visible.

Author response image 6
Individual ASL pH measurements in S0859-treated primary hAECs show that forskolin (FSK) only induced a transient acidification, before the subsequent alkalinisation, in 2 out of 9 independent experiments (panels D and E).

Note that the peaks observed at the time of addition of the forskolin are due to the drop in CO2 when the plates were removed from the plate-reader in order to treat the cells.

3. Figure 2B, D, F: Please define what the symbols represent. Is it pH at a given (which?) timepoint following drug addition? Or mean pH observed over a given (which?) time interval following drug addition?

We have added the following sentence to the figure legend as well as in the methods section. Lane 386-387:

“Changes in ASL pH (ΔASLpH) were calculated by averaging five time points (average pH over 25 min) before and 2hrs after the addition of the molecules (Fsk/S0859)”

4. Figure 2B, D, F: The plotted significance values are confusing. If the error bars on the black and blue dot represent S.E.M., than the difference is unlikely to be significant in either panel. Was the significance calculated for the δ-pH values? If so, it would be helpful to also provide mean+/-S.E.M. δ-pH, and indicate the significance value there.

The statistical analysis was performed on ASL pH (panels B and F) and ΔASLpH (panel D) values. Paired t-tests were chosen as the changes in ASL pH were dependent on the donor cells and passage number. We provide Author response table 1, the raw data and statistical analysis obtained using GraphPad Prism.

Author response table 1
Figure 2 panel BBaselineS0859Figure 2 panel DFSKS0859 + FSKFigure 2 panel FFSKFSK + S0859
20170602 Donor1 P26,526,526,466,4620170602 Donor1 P20,0660,0660,0340,03420170602 Donor1 P26,726,726,686,68
20170607 Donor 1 P46,846,846,786,7820170607 Donor 1 P40,0920,0920,1000,10020170607 Donor 1 P46,866,866,846,84
20170620 Donor 2 P27,137,137,017,0120170620 Donor 2 P20,0550,055-0,029-0,02920170620 Donor 2 P27,287,287,257,25
20170621 Donor 3 P26,946,946,866,8620170621 Donor 3 P20,0900,090-0,001-0,00120170621 Donor 3 P27,127,127,107,10
20170627 Donor 2 P37,227,227,227,2220170627 Donor 2 P30,1250,1250,0330,03320170627 Donor 2 P37,357,357,247,24
20170628 Donor 3P36,626,626,566,5620170628 Donor 3P30,1250,1250,0440,04420170628 Donor 3P36,776,776,706,70
201906057,027,026,936,93201906050,1410,1410,0740,074201906057,287,287,217,21
201906186,876,876,676,67201906180,0770,077-0,030-0,030201906187,097,097,037,03
201906206,436,436,386,38201906200,0790,079-0,013-0,013201906206,526,526,476,47
Table Analyzedmeans S0859 ASL pH baselineTable Analyzedmeans S0859 ASL pH FskTable Analyzedmeans ASL pH Fsk S0859
Column CS0859Column CS0859 + FSKColumn CFSK + S0859
vs.vs,vs.vs,vs.vs,
Column BBaselineColumn BFSKColumn BFSK
Paired t testPaired t testPaired t test
P value0,003P value<0,001P value<0,001
P value summary**P value summary***P value summary***
Significantly different (P < 0.05)?YesSignificantly different (P < 0.05)?YesSignificantly different (P < 0.05)?Yes
One- or two-tailed P value?Two-tailedOne- or two-tailed P value?Two-tailedOne- or two-tailed P value?Two-tailed
t, dft=4,273, df=8t, dft=5,832, df=8t, dft=5,217, df=8
Number of pairs9Number of pairs9Number of pairs9
How big is the difference?How big is the difference?How big is the difference?
Mean of differences (C – B)-0,07895Mean of differences (C – B)-0,07089Mean of differences (C – B)-0,05307
SD of differences0,05543SD of differences0,03647SD of differences0,03052
SEM of differences0,01848SEM of differences0,01216SEM of differences0,01017
95% confidence interval-0,1216 to -0,0363495% confidence interval-0,09893 to -0,0428695% confidence interval-0,07653 to -0,02961
R squared (partial eta squared)0,6954R squared (partial eta squared)0,8096R squared (partial eta squared)0,7728
How effective was the pairing?How effective was the pairing?How effective was the pairing?
Correlation coefficient (r)0,9794Correlation coefficient (r)0,6002Correlation coefficient (r)0,9949
P value (one tailed)<0,001P value (one tailed)0,044P value (one tailed)<0,001
P value summary****P value summary*P value summary****
Was the pairing significantly effective?YesWas the pairing significantly effective?YesWas the pairing significantly effective?Yes

5. Figure 3C: mean+/-S.E.M. bars are hidden behing the individual data points.

Individual points were reduced in size and S.E.M. lines width increased.

6. Figure 3L-M: in panel L please show the data also for the third bar in panel M.

Done.

7. Suppl. Figure 3C, F: It is unclear what the bars represent. E.g., in panel C the two bars seem identical (negative), although in panels A-B S0859 induces a positive current change when added after Fsk (i.e., δ-Isc should be positive for S0859). The same applies for the 2nd and 3rd bars in panel F.

The values in Figure 3—figure supplement 2C represent the negative current elicited after IBMX/FSK and then the amount of such negative current inhibited by S0859. Is the same logic as is plotted in Figure 3C for the amiloride-sensitive current that reflects the inhibition of Na+absorption, or in both cases, the amount of the negative current that is inhibited by the blockers.

8. Suppl. Figure 3G: Several clarifications are needed here:

(i) The legend says "UTP-induced intracellular acidification" but the trace seems to show S0859-induced acidification.

The whole legend is “UTP-induced intracellular acidification in the presence of 30 µM S0859”, and we aim to show the effect of SLC4A4 blocking in UTP-induced intracellular acidification.

(ii) The legend says "in bicarbonate buffer or HEPES", but in the trace it is not indicated where the buffer change has occurred.

The reference to HEPES was a mistake of due to copy and paste from other figures and was taken out from the legend.

(iii) Please calibrate the ordinate into pH units for a better comparison with panel H.

Done.

Reviewer #2 (Recommendations for the authors):

1. SLC4A4, A5 and A8 are the major NCBTs expressed in the primary hAECs (Figure S1A). In my view the arguments used to exclude A5 and A8 as candidate HCO3- importers (l. 298-301) are weak because (i) the fact that transepithelial HCO3- transport generates a Isc does not prove that the importer is electrogenic (cf. Cl- secretory currents mediated through the electroneutral NKCC1 importer), and (ii) the localization of SLC4A5 in the apical membrane of renal epithelial cells does not rule out a basolateral localization in airway cells (cf. the opposite polarization of AE2 in intestinal vs. bile duct cells and of CA12 in salivary duct vs. airway surface cells). Does the SLC4A4 inhibitor S0859, at 30 μm concentration, also inhibit A5 or A8? Are other candidate HCO3- transporters or channels , e.g. Bestrophins, expressed too in primary hAECs?

Answered in the essential revisions section

2. Figure 1A-D: The size of the S0859-inhibitable Isc (representing a HCO3- secretory current) in the hAECs is very small (~1 uAmp/cm2) in comparison with anion current measurements in similar cell models reported previously (e.g. refs. 14, 27: ~20 uAmp/cm2). This is remarkable because the conditions used (Cl- free buffer) is optimal for de-inhibition of NBCe1-B activity that is known to act as a Cli sensor through its 2 GXXXP motifs (Shcheynikov et al. 2015 PNAS 112: E329-37). To judge better about the quality of the hAEC monolayers in culture, one would like to know how the inclusion of Cl-, or CFTR activation by forskolin (affecting the ASL pH; Figure 2) in the perfusion medium affects Isc measurements in the Ussing chamber, and to what extent S0859 inhibits forskolin-stimulated HCO3- currents.

Answered in the essential revisions section

3. Figure 2, G-J: In the hAEC monolayers SLC4A4 seems to be co-expressed with acetylated tubulin in ciliated cells. However it is unclear how many other cell types (CC10-positive Club cells; ionocytes; goblet cells) are retrieved in these human airway cell cultures. In both mouse bronchi and bronchioles (Suppl. Figure 4C) and in differentiated hAECs in the Welsh lab (ref. 15) CFTR (and pendrin) are expressed mainly in CC10+-secretory (Club) cells, not in ciliated cells. This raises the question whether SLC4A4 is also expressed in human Club cells and whether Club cells rather than ciliated cells are the major sources of HCO3- secreted into the ASL.

Answered in the essential revisions section.

4. Figure 3 A-C: Can the authors explain why the IBMX/forskolin/cAMP-induced anion current, in contrast to the UTP/ca2+-induced current, is not reduced in bicarbonate-free buffer, i.e. does not seem to have a HCO3- component? In mouse trachea, most if not all Fsk/cAMP-induced anion secretory current is CaCC-mediated, so there is a priori no reason why forskolin-induced currents would not be reduced (at least in part) in a HCO3- free buffer.

Answered in the essential revisions section.

5. Figure S3D-F: Assuming that the CaCC inhibitor AO1 completely blocks CaCCs and the IBMX/Fsk-induced Isc, how do the authors explain that subsequent addition of S0859 further inhibits a "basal" HCO3- secretory current? Does HCO3- exits the cell at the apical side through tonically active CFTR, or through an electrogenic HCO3-/Cl- exchanger (but pendrin is electroneutral)?

Answered in the essential revisions section

6. Figure 3, D-E, Figure S3A and D, and l. 350: The cause of the transient negative change in Isc elicited by S0859 remains to be identified. Could it be that the compound has transient off-target effects on the intracellular ca2+ level, or activates basolateral K+ channels? It is remarkable that similar deflections are not seen in human AECs (Figure 1C).

Answered in the essential revisions section

7. How close do the hAECs used in this study recapitulate the airways in situ? For example, are CA12 (cf. ref. 11) or ATP12A (cf. ref. 7) highly expressed in these cultures?

8. The systemic Slc4a4 null-mouse model used in this study is known to suffer from many abnormalities, including severe metabolic acidosis due to pRTA (l. 471-480). Whether the reduced level of plasma HCO3- contributes to the lack of HCO3- secretion and reduced ASL pH in the trachea of the Slc4a4-/- mouse (l. 475) depends on the affinity of the NBCe1 transporter for HCO3- ions. Therefore it is of interest to learn whether the transporter is already saturated at 5 mM HCO3-, or needs higher serosal HCO3- levels, e.g. 24 mM.

Answered in the essential revisions section.

9. L. 496: I agree that an airway-specific Slc4a4 null mouse model created by Cre-Lox technology would be of further help in studying the pathogenesis of muco-obstructive diseases of the lungs. In particular a comparison of Club cell-specific vs. ciliated cell-specific Slc4a4-/- mouse models would be useful to study the functional importance of SLC4A4 in each cell type in vivo.

Answered in the essential revisions section.

Reviewer #3 (Recommendations for the authors):

1. The authors show some nice amplification of SLC4A bands and report that both SLC4A7 and SLC4A10 had very low expression levels, referring the reader to Figure S1A. However, it looks as if the expression of SLC4A7 could be quite reasonable in this figure. How did the authors determine relative mRNA abundance and, along similar lines, were the amplified bands digested to confirm their identity?

We have answered this question in the Essential Revisions section, and have amended the text and further discussed these results.

2. The authors provide evidence supporting the expression of several members of the SLC4A family of transporters in human airway epithelial cells. Although S0859 is a blocker of SLC4A4, is it selective for SLC4A4 or is it possible other members of the SLC4A family might be blocked by this drug and contributing to some of the observed effects e.g. ASL pH changes? Can the authors comment on this?

This is now discussed in lines 228-231.

3. The authors show changes in ASL pH in hAEC cultures. Could the authors include in their Methods a statement about the time point at which the pH was considered to have settled, i.e. at what time were the Figure 2B data points collected following the addition of S0859 or Fsk? Could the authors also comment on the difference in timescale between the effects on intracellular acidification (Figure 1) vs the effect on ASL pH which seems to take ~2hrs (Figure 2)?

Details about the ASL pH measurements and analysis have been added to the Methods section (lines 386-391) and in the legend of Figure 2

4. It looks as if some effects are not fully reversible (Figure S3G)? Could the authors comment on this?

Answered in the Essential Revisions section.

5. Direct measurements of ASL volume and pH in the knockout mice would clearly strengthen part of the central thesis, although these measurements are admittedly difficult to perform. However, on a related topic, is there any evidence of changes to mucus rheology in the hAEC cultures accompanying the changes in pH?

Answered in the Essential Revisions section.

https://doi.org/10.7554/eLife.75871.sa2

Article and author information

Author details

  1. Vinciane Saint-Criq

    Biosciences Institute, The Medical School, Newcastle University, Newcastle upon Tyne, United Kingdom
    Present address
    Université Paris-Saclay, INRAE, AgroParisTech, Micalis Institute, Jouy-en-Josas, France
    Contribution
    Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review and editing
    Competing interests
    No competing interests declared
  2. Anita Guequén

    1. Centro de Estudios Científicos, Valdivia, Chile
    2. Universidad Austral de Chile, Valdivia, Chile
    Contribution
    Data curation, Formal analysis, Investigation, Methodology, Writing – review and editing
    Competing interests
    No competing interests declared
  3. Amber R Philp

    1. Centro de Estudios Científicos, Valdivia, Chile
    2. Universidad Austral de Chile, Valdivia, Chile
    Contribution
    Data curation, Investigation
    Competing interests
    No competing interests declared
  4. Sandra Villanueva

    Centro de Estudios Científicos, Valdivia, Chile
    Contribution
    Data curation, Formal analysis, Investigation, Methodology
    Competing interests
    No competing interests declared
  5. Tábata Apablaza

    1. Centro de Estudios Científicos, Valdivia, Chile
    2. Universidad Austral de Chile, Valdivia, Chile
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  6. Ignacio Fernández-Moncada

    Centro de Estudios Científicos, Valdivia, Chile
    Contribution
    Data curation, Formal analysis, Investigation, Methodology
    Competing interests
    No competing interests declared
  7. Agustín Mansilla

    1. Centro de Estudios Científicos, Valdivia, Chile
    2. Universidad Austral de Chile, Valdivia, Chile
    Contribution
    Methodology
    Competing interests
    No competing interests declared
  8. Livia Delpiano

    Biosciences Institute, The Medical School, Newcastle University, Newcastle upon Tyne, United Kingdom
    Contribution
    Investigation
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2319-4456
  9. Iván Ruminot

    1. Centro de Estudios Científicos, Valdivia, Chile
    2. Universidad San Sebastián, Valdivia, Chile
    Contribution
    Formal analysis, Investigation, Methodology, Supervision
    Competing interests
    No competing interests declared
  10. Cristian Carrasco

    Subdepartamento de Anatomía Patológica, Hospital Base de Valdivia, Valdivia, Chile
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  11. Michael A Gray

    Biosciences Institute, The Medical School, Newcastle University, Newcastle upon Tyne, United Kingdom
    Contribution
    Conceptualization, Formal analysis, Funding acquisition, Investigation, Supervision, Writing – review and editing
    Competing interests
    No competing interests declared
  12. Carlos A Flores

    1. Centro de Estudios Científicos, Valdivia, Chile
    2. Universidad San Sebastián, Valdivia, Chile
    Contribution
    Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Supervision, Validation, Writing – original draft, Writing – review and editing
    For correspondence
    1. cflores@cecs.cl
    2. carlos.flores@uss.cl
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3813-1909

Funding

Cystic Fibrosis Trust (SRC003)

  • Michael A Gray

Cystic Fibrosis Trust (SRC013)

  • Michael A Gray

Medical Research Council (MC_PC_15030)

  • Michael A Gray

Fondo Nacional de Desarrollo Científico y Tecnológico (1221257)

  • Carlos A Flores

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

This work was supported by two CF Trust Strategic Research Centre grants (SRC003 and SRC013) and a Medical Research Council (MRC) Confidence in Concept grant (MC_PC_15030) and FONDECYT 1221257 (CAF). The Centro de Estudios Científicos (CECs) was funded by the Base Financing Programme of CONICYT, Chile. Cells from Dr. Randell were supported by Cystic Fibrosis Foundation grant (BOUCHE15R0) and NIH grant (P30DK065988). We would like to acknowledge Drs. Scott H Randell and Leslie Fulcher (Marsico Lung Institute, The University of North Carolina at Chapel Hill, United States) for providing human primary airway epithelial cells from the UNC CF Center Tissue Procurement and Cell Culture Core, and Git Chung for providing human kidney RNA sample.

Ethics

Unless otherwise stated, all procedures were performed after mice were deeply anesthetized via i.p. injection of 120 mg/kg ketamine and 16 mg/kg xylazine followed by exsanguination. All experimental procedures were approved by the Centro de Estudios Científicos (CECs) Institutional Animal Care and Use Committee (#2015-02) and are in accordance with relevant guidelines and regulations.

Senior Editor

  1. Richard W Aldrich, The University of Texas at Austin, United States

Reviewing Editor

  1. László Csanády, Semmelweis University, Hungary

Reviewers

  1. László Csanády, Semmelweis University, Hungary
  2. Hugo de Jonge

Publication history

  1. Received: November 25, 2021
  2. Preprint posted: December 16, 2021 (view preprint)
  3. Accepted: May 27, 2022
  4. Accepted Manuscript published: May 30, 2022 (version 1)
  5. Version of Record published: June 7, 2022 (version 2)

Copyright

© 2022, Saint-Criq et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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  1. Vinciane Saint-Criq
  2. Anita Guequén
  3. Amber R Philp
  4. Sandra Villanueva
  5. Tábata Apablaza
  6. Ignacio Fernández-Moncada
  7. Agustín Mansilla
  8. Livia Delpiano
  9. Iván Ruminot
  10. Cristian Carrasco
  11. Michael A Gray
  12. Carlos A Flores
(2022)
Inhibition of the sodium-dependent HCO3- transporter SLC4A4, produces a cystic fibrosis-like airway disease phenotype
eLife 11:e75871.
https://doi.org/10.7554/eLife.75871

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