Christine Walter

Chloé Rafael

Samia Lassad

Stephanie Baron

Amel Salhi

Ph.D Gilles Crambert
email: gilles.crambert@crc.jussieu.fr

Samia Lasaad

Stéphanie Baron

H,K-ATPase type 2 regulates gestational extracellular compartment expansion and blood pressure in mice. Running title: H,K-ATPase type 2 and gestational blood pressure

Keywords:

teaching and research institutions in France or abroad, or from public or private research centers.

Introduction

The gestation is a physiological situation that requires strong adaptation of the cardio-renal systems in order to cope with the demands of the growing fetus. For this purpose, the extracellular volume is increased by 30-50% and is accompanied by a decrease of the plasma osmolality and of the plasma Na + level [START_REF] Durr | Osmoregulation during pregnancy in the rat. Evidence for resetting of the threshold for vasopressin secretion during gestation[END_REF]. Interestingly, under normal (non-gestational) situation, the decrease of these two parameters should trigger the inhibition of the vasopressin (AVP) production and renal water reabsorption in order to secrete fluid. However, during gestation, the sensitivity of osmoreceptors is modified and AVP production is maintained for reduced levels of plasma osmolality [START_REF] Durr | Osmoregulation during pregnancy in the rat. Evidence for resetting of the threshold for vasopressin secretion during gestation[END_REF]. In addition to salt and water, K + is also retained during gestation, in part, through the stimulation of the H,K-ATPase type 2 (HKA2) [START_REF] Salhi | A link between fertility and K+ homeostasis: role of the renal H,K-ATPase type 2[END_REF][START_REF] West | Renal and colonic potassium transporters in the pregnant rat[END_REF]. This electroneutral transporter (4) consists of two subunits, a catalytic α subunit (encoded by the Atp12a gene) that may combined, in heterologeous systems, with different chaperon-like β subunits [START_REF] Crambert | Human nongastric H+-K+-ATPase: transport properties of ATP1al1 assembled with different beta-subunits[END_REF][START_REF] Geering | Intersubunit interactions in human X,K-ATPases: role of membrane domains M9 and M10 in the assembly process and association efficiency of human, nongastric H,K-ATPase alpha subunits (ATP1al1) with known beta subunits[END_REF]. This transporter exhibits pharmacological and transport features common to two closely related P-type ATPases, the Na,K-ATPase and the H,K-ATPase type 1 (for review see, [START_REF] Crambert | H-K-ATPase type 2: relevance for renal physiology and beyond[END_REF]). For instance, HKA2 may transport Na + instead of H + [START_REF] Codina | The colonic H+,K+-ATPase functions as a Na+-dependent K+(NH4+)-ATPase in apical membranes from rat distal colon[END_REF][START_REF] Cougnon | Does the colonic H,K-ATPase also act as an Na,K-ATPase?[END_REF][START_REF] Crambert | Human nongastric H+-K+-ATPase: transport properties of ATP1al1 assembled with different beta-subunits[END_REF] and is sensitive to ouabain [START_REF] Asano | Functional expression of putative H+-K+-ATPase from guinea pig distal colon[END_REF][START_REF] Cougnon | The rat distal colon P-ATPase alpha subunit encodes a ouabain-sensitive H+, K+-ATPase[END_REF][START_REF] Swarts | The non-gastric H,K-ATPase is oligomycin-sensitive and can function as an H+,NH4(+)-ATPase[END_REF] like the Na,K-ATPase and contributes to the renal secretion of salt [START_REF] Edwards | Versatility of NaCl transport mechanisms in the cortical collecting duct[END_REF][START_REF] Morla | The renal cortical collecting duct: a secreting epithelium?[END_REF]. It also transports H + and is sensitive to Schering 28080 [START_REF] Nakamura | Colonic H+-K+-ATPase is induced and mediates increased HCO3-reabsorption in inner medullary collecting duct in potassium depletion[END_REF] as the H,K-ATPase type 1. The expression of the HKA2 is stimulated by progesterone through a nuclear progesterone receptor-dependent pathways, which induces K + retention [START_REF] Elabida | Chronic potassium depletion increases adrenal progesterone production that is necessary for efficient renal retention of potassium[END_REF][START_REF] Grimont | Mapping of sex hormone receptors and their modulators along the nephron of male and female mice[END_REF], a regulation that may be relevant during gestation.

We have previously shown that the absence of the HKA2 impacts the gestation of mice by impeding efficient K + retention [START_REF] Salhi | A link between fertility and K+ homeostasis: role of the renal H,K-ATPase type 2[END_REF]. Indeed, during gestation, the HKA2KO mice display a decrease of the fertility rate, smaller litter size and higher mortality rate. This phenotype can be reversed by a dietary supplementation in K + . Surprisingly, although those mice do not retain K + as efficiently as their WT littermate during gestation, their plasma K + level remains in the normal range [START_REF] Salhi | A link between fertility and K+ homeostasis: role of the renal H,K-ATPase type 2[END_REF]. We have, therefore, drawn and tested the hypothesis that this normal plasma K + level may be the result of an altered extracellular volume expansion during gestation of HKA2KO mice that would compensate for a putative development of hypokalemia. Therefore we will focus on the regulation of the Na transport systems known to be involved in plasma volume expansion during gestation Thus, we will study the electrogenic transport system of Na + present in the principal cells of the collecting duct mediated by the amiloride-sensitive Na channel (ENaC), the electroneutral ones present in the distal convoluted tubules cells (mediated by the Na + /Cl --exchanger, NCC) and in the β-intercalated cells (mediated by the couple pendrin and the Na + -driven Cl -/HCO 3 -exchanger, NDCBE) and the transport of water by the aquaporin 2 (AQP2).

Material and Methods

Animals

Experiments were performed on C57BL/6J wild-type and knock-out mice for the HKA2 α subunit gene [START_REF] Meneton | Increased sensitivity to K+ deprivation in colonic H,K-ATPase-deficient mice[END_REF]. ). Female WT and HKA2KO were placed for a night with a WT isolated male. The observation of a vaginal plug the next morning confirms the mating. The weight of females is then followed every day to confirm the pregnancy (P). Non-pregnant female mice (not mated with males, NP) served as control for all measured parameters.

Physiological measurements

To record physiological parameters, NP and pregnant (day 16, P) mice were placed in metabolic cages (Techniplast, France) and were fed a standard laboratory diet (0.3 % Na + and 0.6 % K + ; Safe France for more information regarding the detailed composition see http://www.safe-diets.com/wp-content/uploads/2018/01/DS-SAFE-A04.pdf). In mice the gestation lasts 18-20 days, we have therefore chosen day 16 as a representative time point of the late phase of the pregnancy. Urinary creatinine concentrations were determined using an automatic analyzer (Konelab 20i; Thermo, Cergy Pontoise, France). Urinary Na + concentration was determined by flame photometry (IL943, Instruments laboratory, France) and plasma parameters (plasma Na + and hematocrit) were measured by retro-orbital puncture on the anesthetized animal with an ABL77 pH/blood-gas analyzer (Radiometer, Lyon, France). Thiazide (HCTZ) and amiloride-sensitive natriuresis were measured as previously described [START_REF] Leviel | The Na+-dependent chloride-bicarbonate exchanger SLC4A8 mediates an electroneutral Na+ reabsorption process in the renal cortical collecting ducts of mice[END_REF][START_REF] Morla | Renal proteinase-activated receptor 2, a new actor in the control of blood pressure and plasma potassium level[END_REF]. Briefly, the diuretics were administered intraperitoneally in conscious animals placed in metabolic cages (HCTZ: 50mg/kg for 6h of urine collection and amiloride: 5mg/kg for 4h of urine collection). Plasma volume was determined by Evans blue dye dilution as previously described [START_REF] Barron | Role of volume in the regulation of vasopressin secretion during pregnancy in the rat[END_REF][START_REF] Cole | Mice lacking endothelial ACE: normal blood pressure with elevated angiotensin II[END_REF][START_REF] Morgan | Hypervolemia of pregnancy is not maintained in mice chronically overexpressing angiotensinogen[END_REF] in anesthetized animals (a mix of xylazine, 10mg/kg and ketamine 100mg/kg). Plasma osmolality was measured using an osmometer (Vogel, Bioblock Scientific, Strasbourg, FR). Urine aldosterone concentration was determined with a chemiluminescent immunoassay (Diasorin®, Salluggia, Italy) after 24 hours pH 1 acid hydrolysis. Blood pressure was measured in conscious restrained mice by a tail-cuffed plethysmography method (BP2000, Visitech system, France) after a week of adaptation to the apparatus between 9:00 and 11:00 AM before mating. We have chosen the tail-cuffed technic instead of the telemetry because it is less invasive and do not require to anesthetize the pregnant mice to implant the probe. After these measurements, non-pregnant and pregnant females were sacrificed for tissue and organ removal.

Quantitative PCR

RNAs were extracted from whole kidneys using the TRI reagent (Invitrogen, Villebon sur Yvette, France) following the manufacturer's instructions. One µg of total RNA was then reverse-transcribed using the first strand cDNA synthesis kit for RT-PCR (Roche Diagnostics, France) according to the manufacturer's instructions. Real-time PCRs were performed on a LightCycler (Roche Diagnostics, France). No signal was detected in samples that did not undergo reverse transcription or in blank runs without cDNA. In each run, a standard curve was obtained using serial dilution of stock cDNA prepared from mouse kidney total RNA.

The expression of the

Localization of ENaC, NCC and AQP2 on kidney slices

In virgin (NP) or at day 16 of pregnancy (P), anesthetized mice (10mg/kg xylazine and 100mg/kg ketamine) were perfused with 4% paraformaldehyde in the aorta and the kidneys were removed and frozen in optimal cutting temperature compound (VWR). Five µm thick slices were then processed for immunofluorescence microscopy using either an anti-ENaCγ antibody (1/400, StressMarq Biosciences) or an anti-NCC antibody (1/400, StressMarq Biosciences) or an anti-AQP2 (1/400, Santa Cruz). For each antibody, four samples (WT NP, WT P, HKA2KO NP and HKA2KO P, 3 mice/group) were positioned on the same glass slide and treated simultaneously to permit direct comparison of signal intensity. For all slides, the pictures were taken with the same parameters of exposition and magnification (x20).

Organization of the experimental procedures and statistical analysis

The groups of animals that have been used for expression analysis of transporters (PCR, Western blot or immunofluorescence) have not been submitted to any pharmacological treatments (thiazide or amiloride) or blood pressure measurement, have not been punctionned

for blood collection (plasma parameters) or used for plasma volume measurement (Blue Evans) to avoid any possible side effects of these procedures on the results of protein or gene expression. The groups of animals used for plasma volume measurement (Blue Evans) did not undergo other procedures. BP measurements were carried out on groups of animals that did not undergo other procedures (blood collection, metabolic cages, pharmacological treatment).

Results are shown as mean ± s.e.m. Data were tested for significance using two-way ANOVA test followed by a Bonferroni's multiple comparisons post-test or Student's test where appropriate (Prism Software). Outliers were tested by ROUT analysis (Prism Software).

Results

Gestation-induced extracellular volume expansion is blunted in HKA2KO mice

The bodyweight of the non-pregnant NP and pregnant P WT and HKA2KO mice are similar (Table 1) and the number of fetus observed after sacrifice of pregnant females at day 16 postcoïtus is not significantly different between both genotypes (Table1). In WT mice, the late phase of gestation is characterized by a decrease of plasma osmolality (331±14 vs 296±5 mOsm/kg, p=0.038 in non-pregnant NP and pregnant P WT mice, respectively Figure 1A

ENaC expression is differentially regulated in WT and HKA2KO mice during gestation

To understand why the HKA2KO mice did not expand their plasma volume during gestation as the WT mice did, we analyzed the expression of different renal ion transporters known to participate in the salt homeostasis by qPCR (normalized by the expression of rps15, Figure 2A), by Western blot and immunolabelling. ENaC, mediating the electrogenic Na + reabsorption in the collecting duct is composed of three subunits (α, β and γ). As shown in Figures 2B, the expression of ENaCα is stimulated (by 40%, p<0.05) during gestation in kidney of pregnant WT mice (circles) but that of the HKA2KO remains similar before and during gestation (squares). Consequently, the expression level of ENaCα is 30% higher in pregnant WT than in pregnant HKA2KO (p<0.05). As for the β subunit of ENaC, its mRNA expression is not modified by pregnancy and is similar in both genotypes (Figure 2C). The ENaCγ subunit expression only tends to increase during pregnancy in WT mice (Figure 2D, circles, not significant) but is significantly (p<0.05) decreased by 50% in pregnant vs non-pregnant HKAKO mice. Thus, the level expression of ENaCγ subunit is also much lower in pregnant HKA2KO than in pregnant WT (p<0.05). As shown in Figure 2E, although urine aldosterone is increased during pregnancy in both genotypes, it is significantly lower in pregnant HKA2KO mice. When we investigated the protein expression of ENaCα subunit during gestation, we confirmed that it is increased in WT pregnant mice compared to WT non-pregnant mice (Figure 3A, both full-length 90kDa and cleaved 30 kDa bands). In HKA2KO mice, the protein expression of the ENaCα subunit is not modified by pregnancy (Figure 3A). As for the ENaCγ, its protein expression is increased in pregnant WT compared to non-pregnant WT mice (Figure 3B-C) but is not modified by pregnancy in HKA2KO mice (Figure 3B-C).

NCC, pendrin and NDCBE expression during gestation of WT and HKA2KO mice

In addition to ENaC, the distal nephron also expresses two thiazide-sensitive systems of Na + reabsorption, NCC in the distal convoluted tubule (DCT) and the pendrin/NDCBE couple in the β-intercalated cell of the collecting duct. During gestation of WT mice, mRNA expression of NCC is decreased by 50% (Figure 4A, circles, p<0.05) whereas it remains stable in HKA2KO mice (squares, Figure 4A). The mRNA expression of pendrin is strongly increased in pregnant WT (Figure 4B, circles, p<0.01) whereas it remains unchanged in HKA2KO mice.

As for NDCBE, its mRNA expression is not significantly modified during gestation in any genotypes (Figure 4C). At the protein level the results are in good agreement with the observed change at the mRNA level since total NCC expression decreased by 50% during gestation in WT mice but remains unchanged in HKA2KO mice (Figure 4D-E). Protein expression of pendrin is also strongly increased by pregnancy in WT mice and remained unchanged in HKA2KO mice (Figure 4F).

AQP2 expression is not differentially regulated in WT and HKA2KO mice during gestation

We tested the possibility that the water transport system mediated by AQP2 could also be altered during pregnancy in HKA2KO compared to WT mice. As shown in Figure 5, the expression of AQP2 (A and B) and its localization (C) in the principal cells of the collecting ducts are similar in both genotypes whatever the gestational status.

HKA2KO mice exhibit urine salt loss and hypotension during pregnancy

As shown before [START_REF] Salhi | A link between fertility and K+ homeostasis: role of the renal H,K-ATPase type 2[END_REF] , WT or HKA2KO mice increase their food intake by 15-20% during gestation (Figure 6A, p<0.05). However, in parallel, the renal excretion of Na + is not increased (Figure 6B) in the same proportion in pregnant WT mice, characterizing the gestation-induced renal Na + retention. Conversely, the renal excretion of Na + (Figure 6B) is increased in pregnant HKA2KO mice (p<0.05), matching their food intake and revealing that they do not retain Na + as the WT did and therefore do not adapt to the context of gestation.

The Na + retention induced by pregnancy in WT mice is correlated with an increased sensitivity of Na + excretion to amiloride (by 50%, p<0.05, Figure 6C, circles), a result, which is also in a good agreement with the increased expression of ENaC in WT pregnant mice

(

HKA2KO mice exhibit a decreased blood pressure level during pregnancy

In non-pregnant WT and HKA2KO mice, the BP is similar (117.6 vs 114.1 mm Hg, Figure 7 day 0) but the inability of HKA2KO mice to expand their plasma volume during gestation compared to WT mice (because of their renal loss of Na + ) negatively impacts their blood pressure (BP). Thus, in WT (circles) and HKA2KO mice (squares), the BP is significantly modified during gestation. Indeed, as shown in Figure 7, the BP of pregnant WT mice remained similar to that measured before pregnancy (day 0) as already reported [START_REF] Doridot | Preeclampsia-like symptoms induced in mice by fetoplacental expression of STOX1 are reversed by aspirin treatment[END_REF][START_REF] Todkar | Aldosterone deficiency adversely affects pregnancy outcome in mice[END_REF].

However, the BP of HKA2KO mice decreased during the middle phase of gestation and remained 10 mm of Hg lower than that of WT mice.

DISCUSSION

The ability to retain K + during gestation through the activation of the HKA2 is of crucial importance since the absence of this transporter disturbs the process of fertility [START_REF] Salhi | A link between fertility and K+ homeostasis: role of the renal H,K-ATPase type 2[END_REF]. During gestation, the HKA2KO mice exhibited a slight renal and strong intestinal loss of K + resulting in the decrease of litter size, however, without any impact on the plasma [K + ] level [START_REF] Salhi | A link between fertility and K+ homeostasis: role of the renal H,K-ATPase type 2[END_REF]. This was intriguing since in parallel, gestation is a physiological situation characterized by a plasma volume expansion that should lead to a dilution of the plasma components if they are not efficiently retained. We therefore hypothesized that the gestational volume expansion that occurs normally in WT mice could be attenuated in HKA2KO mice.

Limiting plasma volume expansion to avoid the development of hypokalemia?

As shown before [START_REF] West | Increased renal alpha-epithelial sodium channel (ENAC) protein and increased ENAC activity in normal pregnancy[END_REF][START_REF] West | Renal NCC is unchanged in the midpregnant rat and decreased in the late pregnant rat despite avid renal Na+ retention[END_REF], the renal adaptation of the WT mice and rats to pregnancy involves the stimulation of Na + reabsorbing pathways in the collecting duct (ENaC and pendrin) and the downregulation of NCC expression. This situation, somehow, resembles at the mechanisms observed to excrete a load of K + , but in WT pregnant mice or rats, this process is counterbalanced by the stimulation of the HKA2 [START_REF] Salhi | A link between fertility and K+ homeostasis: role of the renal H,K-ATPase type 2[END_REF][START_REF] West | Renal and colonic potassium transporters in the pregnant rat[END_REF], allowing K + to be retained even when ENaC is stimulated. Interestingly, in the absence of HKA2, the lack of K + reabsorption should have resulted to a decrease of plasma [K + ], but instead, we observed that it is the Na + and fluid reabsorption, and in fine, the size of the plasma compartment that is affected. In the absence of the HKA2, the mice are in a paradoxical situation since they need to reduce K loss by decreasing aldosterone production and ENaC activity whereas the increase of the vascular compartment size requires a stimulation of these systems to fill it in. This explains the limitation of the aldosterone production that we observed and is well correlated with the similar gestational phenotype observed in aldosterone synthase KO mice [START_REF] Todkar | Aldosterone deficiency adversely affects pregnancy outcome in mice[END_REF] or in pregnant rats treated with benzamil [START_REF] West | Renal epithelial sodium channel is critical for blood pressure maintenance and sodium balance in the normal late pregnant rat[END_REF] in which the extracellular volume expansion is also blunted.

This crosstalk between the processes controlling the plasma [K + ] and the volemia has already been observed in order to protect male mice under dietary K + restriction against the development of hypokalemia [START_REF] Walter | H,K-ATPase type 2 contributes to salt-sensitive hypertension induced by K+ restriction[END_REF]. Indeed, in the absence of the HKA2 male mice under low-K + diet for a short period of time display a renal loss of Na + and fluid that has been partially attributed to a blockade of the vasopressin action. This eventually contributes to a reduction of the plasma compartment size (by 20%) and lead to hypotension. The water transport system of the collecting duct, based on AQP2 expression analysis, does not seem disturbed neither in normal pregnancy of WT mice, which is different of what has been observed in rat [START_REF] Ohara | Upregulation of aquaporin 2 water channel expression in pregnant rats[END_REF] nor in HKA2KO mice. Here, our results rather suggest that in pregnant mice, the lack of HKA2 contributes to a renal loss of fluid through the inappropriate regulation of ENaC (which is not stimulated in pregnant HKA2KO mice). The mechanisms by which this blunted stimulation of ENaC takes place in HKA2KO mice are not known but they may be related to preservation of the plasma [K + ], avoiding an excess of K + secretion trough the principal cells of the collecting duct. In parallel, this impaired stimulation of ENaC would also result in an inappropriate Na + retention impeding the normal extracellular volume expansion. Since the HKA2 is strongly expressed in colon, we cannot exclude that the effects observed in the KO mice are linked to a fecal loss of K + . However, whatever the origin of the K + balance disturbance, it requires a renal adaptation that leads to the observed gestational phenotypes.

We propose that these two effects protect the organism against the development of hypokalemia, by limiting the K + loss in the urine and reducing the plasma volume. Indeed, if the HKA2KO mice expand their plasma volume as their WT littermate do, their plasma [K + ] level should decrease by 25%, which would result to a severe hypokalemia.

Is HKA2 a putative target to deal with blood pressure modification during the pregnancy ?

The HKA2 therefore appears to be at the crossroad between the regulation of K + , Na + and water metabolism. Its absence during pregnancy leads to compensatory mechanisms that tend to preserve plasma [K + ] level but that leads to hypotension. Interestingly, chronic hypotension during gestation is observed in 2-3% of the pregnancies and is associated with a higher rate of threatened abortion, higher occurrence of severe nausea or vomiting and anemia [START_REF] Banhidy | Hypotension in pregnant women: a population-based case-control study of pregnancy complications and birth outcomes[END_REF]. It would be interesting to investigate whether a low blood pressure during gestation could be related to polymorphisms in the Atp12a gene encoding the catalytic subunit of the HKA2.

Conversely, the identification of the HKA2 as a possible target to reduce blood pressure during pregnancy may also be interesting in the context of gestational hypertension and more particularly during preeclampsia. The volemic status of preeclamptic patient remains controversial, perhaps because this disease is heterogeneous with at least two different components, an early-and a late-onset preeclampsia. At least, the late-onset preeclampsia has been correlated with an increase of total body fluid [START_REF] Gyselaers | Body fluid volume homeostasis is abnormal in pregnancies complicated with hypertension and/or poor fetal growth[END_REF] and of the plasma volume [START_REF] Schrier | Peripheral arterial vasodilation hypothesis of sodium and water retention in pregnancy: implications for pathogenesis of preeclampsia-eclampsia[END_REF][START_REF] Stergiotou | Patterns of maternal vascular remodeling and responsiveness in early-versus late-onset preeclampsia[END_REF]. In this context, the inhibition of the HKA2 could be an interesting therapeutic approach to help decreasing BP. The pharmacology of the HKA2 is complicated and has not been fully investigated (for review see ( 9)). However, some data exist in the literature that suggest that the HKA2 may be inhibited by the "proton-pump inhibitors" (PPI) of the omeprazole family [START_REF] Delpiano | Esomeprazole Increases Airway Surface Liquid pH in Primary Cystic Fibrosis Epithelial Cells[END_REF][START_REF] Lameris | Omeprazole enhances the colonic expression of the Mg(2+) transporter TRPM6[END_REF][START_REF] Min | Proton pump inhibitors decrease eotaxin-3/CCL26 expression in patients with chronic rhinosinusitis with nasal polyps: Possible role of the nongastric H,K-ATPase[END_REF][START_REF] Watanabe | Ouabain-sensitive K(+)-ATPase in epithelial cells from guinea pig distal colon[END_REF]. These compounds are well-known to inhibit the H,K-ATPase type 1, which is structurally and functionally closely related to the HKA2. Recently, these compounds have been proposed to reduce the level of blood pressure in a model of preeclamptic mice [START_REF] Onda | Proton Pump Inhibitors Decrease Soluble fms-Like Tyrosine Kinase-1 and Soluble Endoglin Secretion, Decrease Hypertension, and Rescue Endothelial Dysfunction[END_REF]. In this paper, the authors did not formerly identify the target of omeprazole in this pathological context. Interestingly, PPIs were also shown to reduce blood pressure in hypertensive patients [START_REF] Vazquez | Proton-pump inhibitors therapy and blood pressure control[END_REF]. In light of our own results, we propose that omeprazole may inhibit the HKA2 in the preeclamptic, leading to a modification of the K + , Na + and water balances and thus favoring a decrease of plasma volume and of blood pressure. This hypothesis would have to be investigated further.

Perspectives and Significance

The results of this study establish that the correct expansion of the plasma volume during pregnancy requires the presence of an active HKA2. Indeed, the absence of the HKA2 in pregnant mice, which is already known to promote intestinal and renal K + leaks [START_REF] Salhi | A link between fertility and K+ homeostasis: role of the renal H,K-ATPase type 2[END_REF], results in a reduced volemic expansion allowing the concentration of K + in the extracellular compartment to limit the development of hypokalemia. The consequence of this "underfilled" situation is a lowering of the blood pressure during the late phase of the gestation. The HKA2 appears therefore as a key ion transporter in the physiological adaptation to gestation that controls both K + and Na + metabolisms.

AKNOWLEDGMENTS

Physiological analysis have been performed with the help of Gaelle Brideau and Nadia

Frachon from the "platforme d'exploration fonctionnelle du petit animal" of the team "Physiologie Rénale and Tubulopathies" at the Centre de Recherche des Cordeliers. We are grateful for the technical assistance of the CEF crews in the management of our colony of mice. This study was supported by recurrent grants from the Institut National de la Santé et de la Recherche Médicale (INSERM) and from the Centre National de la Recherche Scientifique (CNRS). It was also supported by the Fondation pour la Recherche Médicale (FRM, project DPC20171138949).

FIGURE LEGENDS

  , dots), a decrease of plasma Na + (150.0±0.4 vs 144.6±0.4 mM, p<0.01 in NP and P WT mice, respectively Fig 1B dots) and a decrease of haematocrit (38.9±0.8 vs 36.5±0.4 %, p=0.019 in NP and P WT mice, respectively Figure 1C, dots). To clearly characterize the volume expansion occurring during gestation we measured it directly with the Evans blue dye dilution method and observed that plasma volume is 1.6-fold increased during gestation in WT mice (990±60 µl vs 1592±193 µl, p<0.01 in NP and P WT mice, respectively Figure 1D, dots). All these modifications induced by the gestation are blunted in HKA2KO mice (Figures 1A-C, squares), their plasma osmolality and haematocrit remain stable (compare to NP HKA2KO mice), their plasma Na + significantly decreases (Figure 1B, 146.5±0.5 mM) but less than in WT (p<0.01) and their plasma volume expansion is limited and is only increased by 20% (1012±71 µl vs 1273±61 µl, p<0.01 in NP and P HKA2KO mice Figure 1D, squares).

  Figure6D, the thiazide-sensitive Na + excretion is not modified by pregnancy, neither in WT nor in HKA2KO mice. This suggests that in WT pregnant mice, the downregulation of NCC (Figure4A-D-E), is compensate by the upregulation of the pendrin (Figure4B-F) as recently proposed by West and collaborators[START_REF] West | The chloride-bicarbonate exchanger pendrin is increased in the kidney of the pregnant rat[END_REF] during pregnancy in rat.

Fig. 1 :

 1 Fig. 1: Characteristics of the plasma volume expansion during gestation in WT and HKA2KO mice Plasma osmolality (A), plasma Na + level (B), and haematocrit (C) were measured on the same groups of animals following a retro-orbital puncture. (D) The measurement of the plasma volume by the Evans blue dye dilution technic was performed in separated groups of mice to avoid consequences of retro-orbital puncture on the results. The presented groups are non-pregnant (NP) and pregnant (P) WT (circles) and NP and P HKA2KO mice (squares). Results are shown as the mean ± s.e.m. Numbers in italic = n of mice. Two-way ANOVA test followed by a Bonferroni's multiple comparisons test, (**

Fig. 3 :Fig. 4 :

 34 Fig. 3: Protein expression of ENaC in WT and HKA2KO mice during gestation Protein expression of the ENaCα (A) and ENaCγ (Β) subunits of in NP and P WT and HKA2KO mice (HKA2KO). (C) Immunolabelling of ENaCγ subunit on NP and P WT and HKA2KO mice (HKA2KO). For each antibody, four samples (WT NP, WT P, HKA2KO NP and HKA2KO P) were positioned on the same glass slide and treated simultaneously to permit direct comparison of signal intensity. For all slides, the pictures were taken with the same parameters of exposition and magnification (x40). Scale bars: 20 µm

Fig. 5 :Fig. 6 :

 56 Fig. 5: AQP2 expression is not modified by gestation in WT and HKA2KO mice Expression of AQP2 mRNA (A) and protein (B) in WT (circles) and HKA2KO mice (HKA2 KO, squares) under normal condition (non-pregnant, NP) or at day 16 post-coïtus (pregnant, P). Results are shown as the mean ± s.e.m. Numbers in italic = n of mice. (C) Immunolabelling of AQP2 on NP and P WT and HKA2KO mice (HKA2KO). The kidney slices (WT NP, WT P, HKA2KO NP and HKA2KO P) were positioned on the same glass slide and treated simultaneously to permit direct comparison of signal intensity. For all slides, the pictures were taken with the same parameters of exposition and magnification (x40). Scale bars: 20 µm

Fig. 7 :

 7 Fig. 7: Blood pressure (BP) level during gestation of WT and HKA2KO mice Systolic BP of WT (circles) and HKA2KO mice (squares) under normal condition (NP) or during gestation (day 9 to day 16 post-coïtus). Results are shown as the mean ± s.e.m.Two-way ANOVA test followed by a Bonferroni's multiple comparisons test (** p<0.01; * p<0.05). BP is impacted by the state of the mice (p<0.01) and by the genotype (p<0.01) but not by the interaction of both.

  Figure2

  Figure 3

  All the animals were kept at CEF (Centre d'Explorations Fonctionnelles of

	the Cordeliers Research Center, Agreement no. A75-06-12). All experiments were conducted
	in accordance with the institutional guidelines and the recommendations for the care and use
	of laboratory animals put forward by the Directive 2010/63/EU revising Directive
	86/609/EEC on the protection of animals used for scientific purposes (project has been
	approved by a user establishment's ethics committee and the Project Authorization : number
	2289.01

Membrane protein extraction and Western Blot Analysis

  Rps15 gene is used to normalize the results. The renal expression of this

	loading accuracy. Western blots were performed according to the standard procedure using a
	polyclonal rabbit anti-ENaCα, anti-ENaCγ, anti-NCC (kindly provided by Jan Loffing,
	University of Zurich, Switzerland) and anti-pendrin (kindly provided by Régine Chambrey,
	Université de la Réunion, France). For quantification, the band intensities were determined
	(ImageJ software) and normalized by Ponceau S red intensity.
	gene is not modified by pregnancy (data not shown). Specific primers for ENaCα (up
	CCAAACGAACCGAACAC;	down	TGTCAGACTTACTCTAGCC),
	ENaCβGGTCCTTATTGATGAGCG; down AGGCGTGAAGTTCCGA), EnaCγ
	(up TCGGTCGTCTGTGTCA; down GCAGATCATCGTCCGTAT), pendrine (up
	CAAAATACCGAGTCAAGGAATGGCT; down CGTTACCGCTGGGCACAAGA), NCC
	(up TCTCACCCTCCTCATCCCCTATCT; down CAGAGCAGCATCCCGAGAGTAATC),
	NDBCE	(up	TTCAACCCATTCAAACTCTTCTATGT;	down
	CCAATGAGCTGAAGCAAGAAA) and rps15 (up TTTCCGAGTAACCGCC; down
	GCAGTGAGTGTTGCTT) transcripts were chosen using the LC Probe design 2.0 software.
	Total kidneys were homogenized in a lysis buffer (250 mM sucrose, 100 mM Tris-Hepes, pH
	7.4 and protease inhibitor cocktail (Complete, Roche Diagnostics)). After removal of
	aggregates and nuclear-associated membrane by low-speed centrifugations, the plasma
	membrane enriched fraction (17000 x g for 30 min) was recovered into the lysis buffer and its
	protein content was measured with the BCA method (Thermo Scientific Pierce). Forty µg of
	protein were then denaturated, resolved by SDS-PAGE (10% polyacrylamide) and transferred
	onto a nitrocellulose membrane. Ponceau red labelling was carried out to check for protein

  -way ANOVA test followed by a Bonferroni's multiple comparisons test, (**

	(NP or P, p=0.357), neither by the genotype (WT or HKA2KO, p=0.912) nor by the
	interaction of both (p=0.368). ENaC γ subunit expression (D) is not impacted by the state
	of the mice (NP or P, p=0.495), neither by the genotype (WT or HKA2KO, p=0.453) but
	by the interaction of both (p=0.0005). Aldosterone level (E) is impacted by the state of
	the mice (NP or P, p<0.01), the genotype (WT or HKA2KO, p=0.014) and by the the
	interaction of both (p=0.026).
	p<0.01; * p<0.05). Plasma osmolality (A) is significantly impacted by the state of the
	mice (NP or P, p=0.0292), by the genotype (WT or HKA2KO, p=0.009) but not by the
	interaction of both (p=0.116). Plasma Na + (B) is significantly impacted by the state of the
	mice (NP or P, p<0.0001), by the genotype (WT or HKA2KO, p=0.004) but not by the
	interaction of both (p=0.067). Hematocrit (C) is significantly impacted by the state of the
	mice (NP or P, p=0.007), not by the genotype (WT or HKA2KO, p=0.694) and by the
	interaction of both (p=0.007). Plasma volume (C) is significantly impacted by the state of
	the mice (NP or P, p<0.0001), by the genotype (WT or HKA2KO, p=0.048) and by the
	interaction of both (p=0.027).
	Fig. 2: Regulation of ENaC mRNA and urine aldosterone level in WT and HKA2KO mice
	during gestation
	Expression of Rps15 (A), ENaC α subunit (B), ENaC β subunit (C), ENaC γ subunit (D)
	mRNAs and urine aldosterone level (E) in WT (circles) and HKA2KO mice (HKA2 KO,
	squares) under normal condition (non-pregnant, NP) or at day 16 post-coïtus (pregnant,
	P). Results are shown as the mean ± s.e.m. Numbers in italic = n of mice. Two-way
	ANOVA test followed by a Bonferroni's multiple comparisons test, (** p<0.01; * p<0.05).
	ENaC α subunit expression (B) is not impacted by the state of the mice (NP or P,
	p=0.2232), neither by the genotype (WT or HKA2KO, p=0.757) but by the interaction of
	both (p=0.0035). ENaC β subunit expression (C) is not impacted by the state of the mice

Table 1 :

 1 General parameters of WT and HKA2KO mice under basal state and during the late part of the gestation. The number of fetus has been calculated after euthanasia of the pregnant females at day 16 post-coïtus and does not obligatory correspond to the number of viable pups.

	7	6	10	8	7	8	10	8
	7	4	6	6	5	6	7	6