key: cord-102498-km03fnc4 authors: Kinaneh, Safa; Knany, Yara; Khoury, Emad; Ismael-Badarneh, Reem; Hammoud, Shadi; Berger, Gidon; Abassi, Zaid; Azzam, Zaher S. title: Identification, Localization and Expression of NHE Isoforms in the Alveolar Epithelial Cells date: 2020-09-03 journal: bioRxiv DOI: 10.1101/2020.09.03.280677 sha: doc_id: 102498 cord_uid: km03fnc4 Na+/H+ exchangers (NHEs), encoded by Solute Carrier 9A (SLC9A) genes in human, are ubiquitous integral membrane ion transporters that mediate the electroneutral exchange of H+ with Na+ or K+. NHEs, found in the kidney and intestine, play a major role in the process of fluid reabsorption together via Na+,K+-ATPase pump and Na+ channels. Nevertheless, the expression pattern of NHE in the lung and its role in alveolar fluid homeostasis has not been addressed. Therefore, we aimed to examine the expression of NHE specific isoforms in alveolar epithelium cells (AECs), and assess their role in congestive heart failure. Three NHE isoforms were identified in AEC and A549 cell line, at the level of protein and mRNA; NHE1, NHE2 and mainly NHE8, the latter was shown to be localized in the apical membrane of AEC. Treating A549 cells with angiotensin (Ang) II for 1 and 3 hours displayed a significant reduction in NHE8 protein abundance and to lesser extent at 5 hours; however, there was no effect at 24 hours. Moreover, A549 treated overnight with Ang II downregulated NHE8 protein abundance. CHF rats held for 1 week had increased abundance of NHE8 compared to sham operated rats. However, lower abundance of NHE8 was observed in CHF rats held for 4 weeks. Herein we show, for the first time, the expression of a novel NHE isoform by AEC, namely NHE8. Besides being negatively affected by Ang II, NHE8 protein levels were distinctly affected in CHF rats, which may be related to CHF severity. Introduction: 51 Alveolar fluid clearance has been shown to be an important mechanism in keeping the 52 airspaces free of edema in both cardiogenic and non-cardiogenic states [1, 2] . There is 53 a large body of evidence that the removal of alveolar fluid is attained by the alveolar 54 epithelial active sodium transport; by which sodium passively enters the alveolar 55 epithelial cells (AEC) via apical amiloride-sensitive Na + channel (ENaC) or other Na + 56 channels and is pumped out of the cells by basolateral Na + , K + -ATPase, an energy 57 consuming process. Following sodium transport, water is extruded from the alveolar 58 airspaces [3] [4] [5] . It has been shown that the survival of acute lung injury patients, 59 directly correlated with the rate of alveolar fluid clearance [6] . 60 The sodium hydrogen exchanger (NHE) family includes several isoforms, which have 61 different characteristics, including cell-compartment localization, plasma membrane 62 distribution and organ-dependent function [7, 8] . 63 The evidence regarding NHE expression and role in the lungs, particularly in alveolar 64 epithelial cells, is scarce. According to the evidence on NHEs in the kidney and 65 intestine; water reabsorption is achieved by the function of Epithelial Na + channel 66 (ENaC), Na + ,K + -ATPase pump along with Na + /H + Exchangers (NHEs) [9] ; thus, it is 67 conceivable to assume that NHE may contribute to this transport in the lung, 68 specifically AEC. Therefore, the major objective of this work is to address whether 69 NHE isoforms are expressed in alveolar epithelial cells and to evaluate their role in 70 alveolar epithelial active sodium transport in healthy and congestive heart failure 71 (CHF) rats. RNA was converted to cDNA using Maxima first strand cDNA kit. The primers used for RT-PCR are listed in Table 1 . cDNA template (10-100ng), Data were presented as mean  SEM; n is the number of animals in each study group. One way analysis of variance was used when multiple comparisons are made 118 followed by a multiple comparison test (Tukey) when the F statistic indicated 119 significance. To analyze paired data, we used unpaired t-test to assess the differences 120 between the study groups. Results were considered significant when p < 0.05. The Kolmogorov-Smirnov test was used to analyze the normality of the groups. The Levene's test was used for comparing the equality of variances. The Student t-test for 123 independent groups was used to compare between two study groups. Two-tailed p 124 value of 0.05 or less was considered to be statistically significant. 128 A549 cells 129 Our major focus was on NHE isoforms that are primarily localized to cell membranes, 130 thus can potentially contribute to the alveolar active sodium transport, and eventually 131 alveolar fluid clearance. Among these are NHE1-5 and NHE8. NHE5, however, was 132 not included in our experiments as it is reported to be exclusively expressed in the 133 brain. By using RT-PCR and targeted primers to each isoform, the expression of 134 NHE1 and NHE8 was confirmed in isolated AEC ( Figure 1A ). Moreover, NHE2 was 135 found to be expressed only following cells incubation for 24 hours ( Figure 1B) ; this 136 observation might be attributed to the differentiation of AEC type II into type I. Similarly, these exchangers, namely NHE1, NHE2 and NHE8 are expressed in A549 138 cell line, known to have characteristic features of AECII (Fig. 1C) . Surprisingly, NHE3 expression was not demonstrated in neither cell types, whereas the expression 140 of unexpected novel isoform, namely NHE8, was established. Based on previous reports, NHE8 might be localized to intracellular compartments or 142 to the plasma membrane. Immunofluorescence staining to NHE8 in AEC and A549 Based on the up mentioned finding of NHE8 abundance in AEC, and its plasma 169 membrane occurring profile, we assume that NHE8 has an important role in alveolar 170 fluid clearance; therefore, we were interested to explore whether its expression is 171 modified in a model of congestive heart failure (CHF) as compared to sham rats. We 172 performed western blot to evaluate NHE8 protein expression in lungs, one (CHF-1w) 173 and four (CHF-4w) weeks following ACF procedure, as compared to sham operated 174 rats. CHF-1w rats exhibited an increased NHE8 protein abundance compared to 8 175 sham-1w rats, yet this elevation was not significant, possibly due to high variability of 176 edema severity among CHF rats (Figures 4 A and C) . However, NHE8 177 immunoreactive levels were significantly decreased in CHF-4w rats as compared to 178 sham 4-w rats (Figures 4 B and D) . This distinct expression suggests that NHE8 179 expression may be related to the severity and progression of CHF. there was no evidence for NHE3 expression in isolated AEC; an observation that was 204 supported by RT-PCR experiments (Fig. 1A) . Therefore, other NHE isoforms known Figure 1B) . Conceivably, this observation may be related to the 213 assumption that NHE2 is localized in AECI. The AECII cell-like cell line, A549, was used to validate NHE isoforms expression (Figures 2A-C) . Western blotting to isolated 227 basolateral membranes of AEC was negative for NHE8 expression (Fig. 2D) . These 228 findings support the assumption that NHE8 is localized on the apical side of AEC. Unfortunately, there is no known selective inhibitor for NHE8 since it is not much 230 studied and only recently has been discovered. Therefore, we decided to bypass this 231 obstacle and study the response of this isoform to hormones and factors known to 232 impair the ability of the lungs to clear edema. . Recently, our group has shown that Ang II impaired AFC, partly by down-237 regulating Na + , K + -ATPase protein levels [12] . Assuming that NHE8 participates in 238 vectorial sodium transport, we hypothesized that Ang II may affect NHE8 protein 239 expression. Therefore, we conducted an experiment in which A549 cells were treated 240 with Ang II (10 -8 M) and examined NHE8 protein changes over different periods of 241 time-1, 3, 5 and 24 hours. As shown in Fig. 3 , Ang II decreased the levels of NHE8 242 mature protein at 1, 3, and 5 hours; while there was no effect following 24 hours of Furthermore, we investigated NHE8 expression in CHF rats, using ACF rat model. Rats were sacrificed 1 week (CHF-1w) or 4 weeks (CHF-4w) following ACF 254 procedure. NHE8 was distinctly expressed in CHF rat lungs, in which CHF-1w NHE8 255 levels were increased, while in CHF-4w, NHE8 levels were decreased, as compared to 256 sham-rats (Fig. 4) . This distinct expression pattern of NHE8 might suggest a 257 protective role of NHE8 in CHF-1w that is driven by the need to clear excessive lung 258 fluids; while the decreased levels in CHF-4w, might be a result of CHF severe 259 condition, were even protective pathways are badly damaged. The main limitation of this study was our inability to directly address NHE8 effect on 261 AFC due to the lack of specific inhibitors or NHE8 knock-out mice models. latter it is localized to the apical membrane. By exchanging extracellular Na + with 379 intracellular H + , NHE8 contribute to Na + vectorial transport along with epithelial 380 sodium channel (ENaC). The entered sodium then is pumped out of alveolar cells by 381 Na + , K + ATpase pump. Sodium transport from alveolar space to the interstitium is 382 accompanied by water movement and so edema clearance. Resolution of pulmonary edema thirty years of progress Patterns of alveolar fluid clearance in heart 280 failure Alveolar epithelium and Na,K-ATPase in acute 282 lung injury Invited review: lung edema clearance: role of 284 K(+)-ATPase Lung Edema Clearance: 20 Years 286 of Progress Invited Review: Alveolar edema fluid clearance in the injured lung Alveolar fluid clearance is impaired in the majority of 289 patients with acute lung injury and the acute respiratory distress syndrome The Na+/H+ exchanger: An update on structure, 292 regulation and cardiac physiology Regulation of myocardial Na+/H+ exchanger activity Structural and functional 297 analysis of the Na+/H+ exchanger Natriuretic peptides system in the pulmonary tissue of rats with heart failure: 301 Potential involvement in lung edema and inflammation Isolation and culture of alveolar type II cells The role of Angiotensin II and cyclic AMP in alveolar active sodium 307 transport Regulation of ion channel structure and function by reactive oxygen-nitrogen 311 species Lung epithelial fluid transport and the 313 resolution of pulmonary edema Role of Na+/H+ exchanger 315 NHE3 in nephron function: Micropuncture studies with S3226, an inhibitor of 316 NHE3 Mechanisms of pulmonary edema clearance Evolutionary origins of eukaryotic 321 sodium/proton exchangers SLC9/NHE gene family, a plasma 324 membrane and organellar family of Na +/H+ exchangers Ontogeny of NHE8 in the rat proximal tubule The neurohormonal hypothesis: a theory to explain the mechanism 332 of disease progression in heart failure A new coronavirus 334 associated with human respiratory disease in China A Call for Rational 337 Intensive Care in the Era of COVID-19