Yohan Bignon

Laurent Pinelli

Nadia Frachon

Olivier Lahuna

Lucile Figueres

Pascal Houillier

Stéphane Lourdel

Jacques Teulon

Marc Paulais
email: marc.paulais@inserm.fr

Defective bicarbonate reabsorption in Kir4.2 potassium channel deficient mice impairs acid-base balance and ammonia excretion

Keywords: Ammoniagenesis, Isolated metabolic acidosis, Kir4.2 potassium channel, Proximal tubule

The kidneys excrete the daily acid load mainly by generating and excreting ammonia but the underlying molecular mechanisms are not fully understood. Here we evaluated the role of the inwardly rectifying potassium channel subunit Kir4.2 (Kcnj15 gene product) in this process. In mice, Kir4.2 was present exclusively at the basolateral membrane of proximal tubular cells and disruption of Kcnj15 caused a hyperchloremic metabolic acidosis associated with a reduced threshold for bicarbonate in the absence of a generalized proximal tubule dysfunction. Urinary ammonium excretion rates in Kcnj15-deleted mice were inappropriate to acidosis under basal and acid-loading conditions, and not related to a failure to acidify urine or a reduced expression of ammonia transporters in the collecting duct. In contrast, the expression of key proteins involved in ammonia metabolism and secretion by proximal cells, namely the glutamine transporter SNAT3, the phosphate-dependent glutaminase and phosphoenolpyruvate carboxykinase enzymes, and the sodium-proton exchanger NHE-3 was inappropriate in Kcnj15deleted mice. Additionally, Kcnj15 deletion depolarized the proximal cell membrane by decreasing the barium-sensitive component of the potassium conductance and caused an intracellular alkalinization. Thus, the Kir4.2 potassium channel subunit is a newly recognized regulator of proximal ammonia metabolism. The kidney consequences of its loss of function in mice support the proposal for KCNJ15 as a molecular basis for human isolated proximal renal tubular acidosis.

TRANSLATIONAL STATEMENT

Chronic metabolic acidosis caused by a failure to adapt renal ammonia excretion to the daily net acid load is an independent risk factor for the progession of the chronic kidney disease towards end-stage renal disease and is significantly associated with higher mortality. We report a new molecular actor in ammonia metabolism whose defect leads to isolated proximal renal tubular acidosis in mice. This study may shed more light on the pathophysiology of this inherited disease and improve our understanding of the mechanisms underlying acid-base regulation in human health and disease; it also may also provide a basis for accurate clinical and genetic diagnosis and future design of targeted therapeutics along with, possibly, strategies for gene therapy of this disorder.

INTRODUCTION

Commonly associated is with chronic kidney disease, metabolic acidosis accelerates the progression of chronic kidney disease to end-stage renal disease and leads to higher mortality. [START_REF] Kraut | Consequences and therapy of the metabolic acidosis of chronic kidney disease[END_REF] Renal ammonia a metabolism is a cornerstone of kidney's ability to excrete acid, and a declining ability to excrete the daily net acid load because a failure to adapt renal ammonia excretion is an independent risk factor for the progession of chronic kidney disease toward end-stage renal disease. [START_REF] Vallet | Urinary ammonia and long-term outcomes in chronic kidney disease[END_REF] (In this article, the term "ammonia" refers to the combination of NH 3 and NH 4 + . The term "NH 3 " or "NH 4 + " is used only when referring to a specific molecular form.) Because most of the excreted ammonia arises from its production and secretion by proximal tubular cells, deciphering the events governing proximal ammonia metabolism is of fundamental importance for comprehending the pathophysiology of disorders involving renal acid excretion.

Information regarding regulation of ammonia metabolism during proximal renal tubular acidosis (pRTA) is limited and mainly arose from clinical studies of patients with permanent forms of familial isolated pRTA. This rare acid-base disturbance, although primarily defined by a failure to reabsorb filtered HCO 3 -in the absence of a generalized proximal tubule dysfunction, is also associated with inappropriately low urinary NH 4 + excretion rates under basal and acidloading conditions. [START_REF] Haque | Proximal renal tubular acidosis: a not so rare disorder of multiple etiologies[END_REF] To date, only the Na + -HCO 3 -cotransporter NBCe1, whose loss of function by mutations in solute carrier family 4 member 4 SLC4A4 causes the most common form of isolated pRTA, has been identified as a critical actor in abnormal ammonia metabolism in this disease. Thus Slc4a4 deletion in mice causes severe pRTA and impairs urinary NH 4 + excretion by reducing the proximal expression of ammoniagenic enzymes. [START_REF] Handlogten | NBCe1 expression is required for normal renal ammonia metabolism[END_REF][START_REF] Lee | NBCe1-A Regulates Proximal Tubule Ammonia Metabolism under Basal Conditions and in Response to Metabolic Acidosis[END_REF][START_REF] Gawenis | Colonic anion secretory defects and metabolic acidosis in mice lacking the NBC1 Na+/HCO3-cotransporter[END_REF] However, the finding that rarer cases of isolated pRTA occur independently of SLC4A4 mutations indicates that other regulators of renal ammonia metabolism may be involved in the reduced NH 4 + excretion in this disease. [START_REF] Katzir | Familial pure proximal renal tubular acidosis--a clinical and genetic study[END_REF] Early studies in mice suggested that the basolateral potassium conductance of proximal tubular cells might be a key player in the regulation of ammonia metabolism. [START_REF] Nagami | Effect of bath and luminal potassium concentration on ammonia production and secretion by mouse proximal tubules perfused in vitro[END_REF][START_REF] Völkl | Electrophysiology of ammonia transport in renal straight proximal tubules[END_REF] Several potassium channel subunits that may underlie this conductance have been dentified on a molecular level, including the inwardly rectifying Kir4.2 (Kcnj15) subunit. [START_REF] Hebert | Molecular diversity and regulation of renal potassium channels[END_REF] Here, we used a new strain of mice in which Kcnj15 is deleted and demonstrate that basolateral Kir4.2 is a major determinant of the membrane potential of proximal cells and regulates ammonia metabolism.

These results provide further insight into the molecular mechanisms involved in renal acid-base homeostasis and support KCNJ15 as a new candidate for yet unelucidated isolated proximal metabolic acidoses.

RESULTS

Validation of mice with deletion of Kcnj15. Adult mice carrying a disrupted Kcnj15

gene were obtained as detailed in the Supplementary Methods section. The successful incorporation of the mutant allele was established by polymerase chain reaction (PCR) on genomic DNA from tail biopsies (Figure 1a), and the absence of native Kir4.2 protein in Kcnj15 -/- mice was confirmed by Western blot analysis of kidneys (Figure 1b), as well as in lung and stomach where Kir4.2 is also present. [START_REF] He | Acid secretion-associated translocation of KCNJ15 in gastric parietal cells[END_REF] In the kidney of Kcnj15 +/+ mice, major Kir4.2-specific bands were observed near the predicted molecular mass of the monomeric peptide [START_REF] Hill | Cloning, expression, and localization of a rat hepatocyte inwardly rectifying potassium channel[END_REF] and corresponding to its glycosylation status as observed by others on mouse renal cortex using the same Kir4.2 antibody. [START_REF] Hibino | Differential assembly of inwardly rectifying K+ channel subunits, Kir4.1 and Kir5.1, in brain astrocytes[END_REF] Cellular distribution and subcellular localization of renal Kir4.2. In accordance with observations in the human kidney, [START_REF] Chabardès-Garonne | A panoramic view of gene expression in the human kidney[END_REF] real-time PCR on glomeruli and tubular segments isolated from kidneys of Kcnj15 +/+ mice revealed Kir4.2 mRNA expression exclusively in the convoluted and straight portions of the proximal tubule (Figure 2a). The Kir4.2 peptide was detected in renal cortical and subcortical regions (Figure 2b) and more specifically in proximal cells of cortex and outer stripe of the outer medulla (Figure 2c). No Kir4.2 labeling was observed in the inner stripe of the outer medulla devoid of proximal tubule (Figure 2d). In cortical cells of Kcnj15 +/+ mice, the Kir4.2 peptide was observed in a region ranging from the basal side up to nearly two thirds of cell height, likely corresponding to basolateral membrane infoldings, leaving unstained a shallow subapical region and microvilli (Figure 3a). The Kir4.2 immunofluorescence intensity profile peaked at 1.6 ± 0.3 µm (n = 13 cells) away from the basal pole (Figure 3b), indicating a basolateral localization.

Phenotype of mice with Kcnj15 deletion. Kcnj15 -/-mice exhibited no excess mortality or detectable growth retardation and were indeed indistinguishable from their Kcnj15 +/+ littermates by gross physical appearance (Supplementary Figure S1). Their daily water and food intakes were normal (P = 0.67 and 0.71, respectively, vs Kcnj15 +/+ mice) and their kidneys showed no alteration in gross external appearance and weight and no morphologic or ultrastructural abnormality (Suplementary Figure S1).

Analyses of plasma and urine of Kcnj15 -/-mice revealed no sign of polyuria, salt wasting, or elevated fractional excretions of phosphate, glucose and proteins; in addition, their normal estimated glomerular filtration rate indicated no chronic renal failure (Tables 1 and2). In contrast, Kcnj15 -/-mice were spontaneously acidotic. Their lower blood HCO 3 -concentration was counterbalanced by an equivalent increase in plasma Cl -concentration (Figure 4a), which, together with normal plasma Na + and K + concentrations (Table 1), yielded a normal plasma anion gap. Kcnj15 -/-mice also spontaneously produced an acidic urine (P < 0.0001 vs Kcnj15 +/+ mice) with a barely significant HCO 3 -wasting (P < 0.04 vs Kcnj15 +/+ mice; Figure 4b). To determine whether this minimal HCO 3 -loss reflected a new steady state where blood HCO 3 -content was lowered to a value close to a reduced renal threshold for HCO 3 -, mice were challenged with 24hour HCO 3 -loads. In these conditions, the HCO 3 -filtered loads in the two groups of mice were similar (Supplementary Table S1) and urinary pH increased because bicarbonaturia occurred when blood HCO 3 -concentration rose above their respective renal threshold for HCO 3 -(Figure 4b). Nevertheless, urine alkalinization and bicarbonaturia in Kcnj15 -/-mice appeared at blood HCO 3 -concentrations that were lower than the low normal range in Kcnj15 +/+ mice, highlighting a reduced renal threshold for HCO 3 -. In the absence of a generalized proximal dysfunction (described previously), Kcnj15 deletion therefore caused an isolated pRTA.

NH 4 + excretion under basal and exogenous acid-loading conditions. Under basal conditions, Kcnj15 -/-mice did not exhibit the expected increase in urinary NH 4 + content in response to acidosis and only slightly increased net acid excretion (Figure 5a), as expected from an acidosis of tubular origin. To determine whether they were able to excrete maximal amounts of NH 4 + , Kcnj15 -/-mice were challenged with a 8-day exogenous NH 4 Cl load. After 2 days, Kcnj15 -/-mice exhibited almost 2-fold greater changes in blood pH and blood HCO 3 -and plasma Cl -concentrations than did Kcnj15 +/+ mice (P < 0.05 for each parameter; Figure 5b), despite a ~ 20 % lower acid load (Supplementary Figure S2), likely due to a reduced water intake (Supplementary Table S2). Then, wheras Kcnj15 +/+ mice completely recovered at day 8 of the acid-loading period, Kcnj15 -/-mice remained acidotic (P < 0.05 vs day 0 for blood pH and for blood HCO 3 -and plasma Cl -concentrations; n = 5-10). All along the treatment period, the urinary NH 4 + excretion rate in Kcnj15 -/-mice progressively increased but remained one-third lower than in Kcnj15 +/+ mice (P < 0.001, analysis of variance; Figure 5c). Actually, Kcnj15 deletion reduced by a factor of ~ 10 the sensitivity of the changes in urinary NH 4 + excretion to the degree of metabolic acidosis (in mmol urinary NH 4 + /mmol creatinin per mM blood HCO 3 -: 117 in Kcnj15 +/+ mice vs 11 in Kcnj15 -/-mice). Also, the urinary pH in Kcnj15 -/-mice remained more acidic than in Kcnj15 +/+ mice all throughout the acid-loading period (Figure 5d), indicating an intact ability to achieve a high urine free H + concentration but not matched by an appropriate NH 4 + excretion.

The mRNA levels for the rhesus proteins RhBG and RhCG, 2 major transporters involved in final urinary ammonia secretion by distal cells, [START_REF] Weiner | Ammonia transport in the kidney by Rhesus glycoproteins[END_REF] were normal in Kcnj15 -/-mice (Supplementary Figure S3). Also, Kcnj15 -/-mice fully concentrated their urine under basal and acid-loading conditions (Table 2 and Supplementary Table S2), indicating an intact medulla, a prerequisite for the accumulation of NH 3 into the interstitium, facilitating its transfer into the collecting duct lumen. [START_REF] Weiner | Role of NH3 and NH4+ transporters in renal acid-base transport[END_REF] These results excluded a low NH 4 + excretion related to a defective distal nephron in Kcnj15 -/-mice and altogether pointed toward a defect in proximal ammoniagenesis.

Expression of enzymes and transporters relevant for ammoniagenesis.

Ammoniagenesis in the proximal tubule arises from the basolateral uptake of glutamine and its metabolism by a series of mitochondrial and cytosolic enzymes, both highly upregulated by metabolic acidosis or an exogenous acid load. [START_REF] Moret | Regulation of renal amino acid transporters during metabolic acidosis[END_REF][START_REF] Schoolwerth | Changes in mRNAs for enzymes of glutamine metabolism in kidney and liver during ammonium chloride acidosis[END_REF] We first performed quantitative reverse transcription PCR on whole kidney preparations for the basolateral transporter system N1 Na + and H + -coupled glutamine transporter (SNAT3), the phosphate-dependent glutaminase (PDG), glutamate dehydrogenase (GDH), and phosphoenolpyruvate carboxykinase (PEPCK) enzymes, and the apical Na + /H + exchanger (NHE-3), the primary pathway for NH 4 + secretion by proximal tubular cells. Compared with Kcnj15 +/+ mice, the basal mRNA abundance of SNAT3 and PEPCK was reduced in Kcnj15 -/-mice, with those of PDG, GDH remaining unchanged (Figure 6). In addition, the response of Kcnj15 -/-mice to the acid load was significantly blunted for SNAT3, GDH and PEPCK and absent for PDG. In contrast, the mRNA for the broad neutral (0) amino acid transporter 1 (B 0 AT1) and L-type amino acid transporter 2 (LAT2) transporters, typically involved in glutamine reabsorption, were only marginally affected by Kcnj15 deletion and the acid load (Supplementary Figure S4). Lastly, NHE-3 mRNA abundance in Kcnj15 -/-mice under basal and acid-loading conditions was similar to that in Kcnj15 +/+ mice.

We substantiated these observations by Western blots experiments; the results are summarized in Figure 7. Under basal condition, PEPCK, PDG and NHE-3 proteins abundance in Kcnj15 -/-mice was in accordance with results on mRNA levels, whereas that of SNAT3 was more elevated than in Kcnj15 +/+ mice, opposite to our mRNA data. In addition, PEPCK peptide expression in acid-loaded Kcnj15 -/-mice followed enzyme mRNA level, whereas that of SNAT3, PDG and NHE-3 did not exhibit the higher protein levels expected from the more severe acidosis in this condition.

Intracellular pH, membrane potential and NBCe1 expression determination. It has

been early suggested that the reduced rate of ammonia excretion in patients with isolated pRTA might originate from an increase in intracellular pH (pH i ), possibly caused by an impaired basolateral exit of HCO 3 -. [START_REF] Halperin | What is the underlying defect in patients with isolated, proximal renal tubular acidosis?[END_REF] As summarized in Figure 8a, the resting pH i of cells bathed in a Na +and HCO 3 --containing solution was significantly higher, that is more alkaline, in Kcnj15 -/-mice (7.26 ± 0.1, n = 6 tubules from 4 mice) than in Kcnj15 +/+ mice (6.95 ± 0.1, n = 6 tubules from 4 mice) (P = 0.036). The blockade of the basolateral Na + -HCO 3 -exit from proximal cells increasing pH i , [START_REF] Matsumura | Regulatory mechanism of cell pH in the renal proximal tubule of bullfrog nephron[END_REF] a decreased expression of NBCe1, responsible for the basolateral exit of HCO 3 -out of proximal tubular cells, [START_REF] Haque | Proximal renal tubular acidosis: a not so rare disorder of multiple etiologies[END_REF] or of the TWIK-related acid-sensitive K + channel 2 (Task2) potassium channel, proposed to sustain the activity of the rheogenic NBCe1, [START_REF] Warth | Proximal renal tubular acidosis in TASK2 K+ channeldeficient mice reveals a mechanism for stabilizing bicarbonate transport[END_REF] could have accounted for the alkaline pH i in Kcnj15 -/-mice. However, Kcnj15 deletion leaved unchanged NBCe1 and Task2 mRNA levels (Supplementary Figure S5) and did not decrease their respective protein abundance, with that of NBCe1 even increasing (Figure 8b).

Because a depolarization of the basolateral membrane of proximal tubular cells, induced by high bath K + concentration or the addition of barium, causes an intracellular alkalinization dependent on basolateral Na + -HCO 3 -transport, [START_REF] Wang | Fused cells of frog proximal tubule: II. Voltagedependent intracellular pH[END_REF][START_REF] Alpern | Mechanism of basolateral membrane H+/OH-/HCO-3 transport in the rat proximal convoluted tubule. A sodium-coupled electrogenic process[END_REF] the impact of Kcnj15 deletion on membrane potential (V m ) was then investigated (Figure 8c). In Kcnj15 +/+ mice, the resting V m was -52 ± 2.5 mV (n = 14 patches from 6 mice), in reasonnable agreement with values measured in mice using microelectrodes, [START_REF] Völkl | Potassium conductance in straight proximal tubule cells of the mouse. Effect of barium, verapamil and quinidine[END_REF][START_REF] Vallon | Role of KCNE1-Dependent K+ Fluxes in Mouse Proximal Tubule[END_REF] and markedly less negative with 1 mM barium in the bath (V m = -21 ± 2.4

mV, n = 6 patches from 4 mice). At rest, the proximal cell membrane of Kcnj15 -/-mice was significantly depolarized (V m -32 ± 3.1 mV ; n = 16 patches from 10 mice) and bath barium had a reduced effect (V m = -21 ± 1.9 mV, n = 6 patches from 4 mice). We next attempted to evaluate the impact of Kcnj15 deletion on the rheogenic NBCe1 activity by using conditions producing changes in pH i whose magnitude and rate constant reflect the basolateral Na + -HCO 3 -cotransport. [START_REF] Boron | Intracellular pH regulation in the renal proximal tubule of the salamander. Basolateral HCO3-transport[END_REF][START_REF] Yoshitomi | Rheogenic sodium-bicarbonate cotransport in the peritubular cell membrane of rat renal proximal tubule[END_REF] Original pH i recordings are shown in Figure 8d, and the detailed kinetics analysis of the changes in pH i is given in Table 3. Stimulating either Na/HCO 3 exit by bath Na + withdrawal or then entry by bath Na + readdition in Kcnj15 +/+ and Kcnj15 -/-mice caused respective initial changes in pH i with statistically similar kinetics, although quite variable from tubule to tubule within a group of mice. Nevertheless, while pH i either remained stable under Na + -free condition (3 tubules, see Figure 8d) or slightly recovered (3 tubules; not shown) in Kcnj15 +/+ mice, it rose at or above its initial resting level (2 tubules, see Figure 8d) or partially recovered (3 tubules; not shown). Therefore, the presence of a pH i regulatory mechanism in Kcnj15 -/-mice, despite the absence of luminal and bath Na + , together with the more alkaline pH i values, precluded further computational analysis of our data in terms of Na/HCO 3 transport rates and NBCe1 activity. (The amplitude and rate constant of the pH i changes cannot be taken from experiments in which pH i regulatory mechanisms are still operating and may lead to underestimates. In addition, the amplitude of the changes in pH i is also inversely proportional to the intracellular buffering power [ which depends on pH i .)

DISCUSSION

The basolateral membrane conductance of mouse proximal cells is almost purely selective to K + and markedly inhibited by barium. [START_REF] Völkl | Potassium conductance in straight proximal tubule cells of the mouse. Effect of barium, verapamil and quinidine[END_REF][START_REF] Völkl | Effects of ouabain and temperature on cell membrane potentials in isolated perfused straight proximal tubules of the mouse kidney[END_REF] Our results show that the inwardly rectifying Kir4.2 subunit underlies almost two thirds of the whole membrane barium-sensitive potassium conductance. In our experimental conditions, luminal potassium channels may have contaminated our membrane potential measurements in the presence of barium, [START_REF] Hebert | Molecular diversity and regulation of renal potassium channels[END_REF] but given that the basolateral membrane potassium conductance is much higher than that of the luminal membrane, [START_REF] Völkl | Potassium conductance in straight proximal tubule cells of the mouse. Effect of barium, verapamil and quinidine[END_REF] such a contamination would be minimal and, in any case, have led to an underestimation of the contribution of Kir4.2 to the basolateral membrane potassium conductance.

A barium-sensitive conductive transport of NH 4 + across the basolateral membrane of mouse proximal cells may contribute to its cellular uptake. [START_REF] Völkl | Electrophysiology of ammonia transport in renal straight proximal tubules[END_REF] Although the permeability of Kir4. channel is only ~ 0.1). [START_REF] Edvinsson | Kir4.1 K+ channels are regulated by external cations[END_REF][START_REF] Weinstein | Ammonia transport in a mathematical model of rat proximal tubule[END_REF] Lastly, the decreased basolateral potassium conductance in the absence of Kir4.2 could have altered Na + /K + -adenosine triphosphatase activity and NH 4 + entry, through its substitution for K + at the K + binding site, [START_REF] Kurtz | Ammonium as a substrate for Na+-K+-ATPase in rabbit proximal tubules[END_REF][START_REF] Garvin | Ammonium replaces potassium in supporting sodium transport by the Na-K-ATPase of renal proximal straight tubules[END_REF] but kinetic models suggest a potentially significant NH 4 + transport by this pathway only in the inner medulla, [START_REF] Weinstein | Ammonia transport in a mathematical model of rat proximal tubule[END_REF][START_REF] Kurtz | Ammonium as a substrate for Na+-K+-ATPase in rabbit proximal tubules[END_REF] devoid of proximal tubule.

The reduced sensitivity of the changes in urinary NH 4 + excretion to the degree of metabolic acidosis in mice with deletion of Kcnj15 was more clearly associated with the inappropriate expression of major ammoniagenic enzymes under basal and acid-loading conditions. Of note, the marked decrease in PEPCK expression would raise intramitochondrial glutamate and -ketoglutarate contents and further decrease PDG and GDH activities by competitive inhibition. [START_REF] Weiner | Renal ammonia metabolism and transport., Renal Ammonia Metabolism and Transport[END_REF] In addition, the inappropriate protein expression of SNAT3 under acidloading condition likely blunted glutamine uptake and availability for ammoniagenesis. Whereas the increased basal expression of SNAT3 would favor glutamine uptake and ammoniagenesis, as expected from spontaneously acidotic mice, 17 SNAT3 transports glutamine with Na + in exchange for H + so that the rise in pH i we observed in mice with deletion of Kcnj15 would decrease the chemical gradient for H + and blunt glutamine uptake. [START_REF] Chaudhry | Molecular analysis of system N suggests novel physiological roles in nitrogen metabolism and synaptic transmission[END_REF] That Kcnj15 deletion also may impair NH 4 + secretion by NHE-3 remains unclear. Because acidosis increases NHE-3 protein expression in cells of proximal tubules and thick ascending limb, [START_REF] Ambühl | Chronic metabolic acidosis increases NHE3 protein abundance in rat kidney[END_REF][START_REF] Laghmani | Chronic metabolic acidosis enhances NHE-3 protein abundance and transport activity in the rat thick ascending limb by increasing NHE-3 mRNA[END_REF] all or part of the inappropriate NHE-3 expression in mice with deletion of Kcnj15 cannot yet be precisely assigned to either nephron segment. In any case, since NHE-3 in thick ascending limbs cells is supposed to secrete NH 4 + , [START_REF] Weiner | Role of NH3 and NH4+ transporters in renal acid-base transport[END_REF] its reduced expression in the proximal tubule and/or in the TAL would ultimately lower the NH 4 + content of the final urine.

In patients with isolated pRTA, the low NH 4 + excretion rates are thought to result from an intracellular alkalinization. [START_REF] Halperin | What is the underlying defect in patients with isolated, proximal renal tubular acidosis?[END_REF] The increased pH i in proximal cells of mice lacking Kir4.2 supports this hypothesis. In patients carrying loss-of-function mutations in SLC4A4, as well as in mice where Slc4a4 deletion impairs urinary NH 4 + excretion by the reduced expression of ammoniagenic enzymes, [START_REF] Handlogten | NBCe1 expression is required for normal renal ammonia metabolism[END_REF][START_REF] Lee | NBCe1-A Regulates Proximal Tubule Ammonia Metabolism under Basal Conditions and in Response to Metabolic Acidosis[END_REF][START_REF] Matsumura | Regulatory mechanism of cell pH in the renal proximal tubule of bullfrog nephron[END_REF] the most straightforward explanation to an elevated pH i would be an intracellular accumulation of HCO 3 -caused by the absence of NBCe1 at the cell membrane. Our observation that Kcnj15 deletion did not decrease NBCe1 protein expression suggests another mechanism. (Acidosis notoriously has no effect on the expression of the major NBCe1-A isoform protein in the cortex but increases the expression of the minor NBCe1-B isoform in the outer stripe of the outer medulla. The increased NBCe1 protein expression we observed thus may reflect the upregulation of NBCe1-B.) Notably, NBCe1 is rheogenic rendering both the magnitude and polarity of basolateral HCO 3 -transport sensitive to changes in membrane potential. [START_REF] Soleimani | Stoichiometry of Na+-HCO-3 cotransport in basolateral membrane vesicles isolated from rabbit renal cortex[END_REF] In particular, a depolarization decreases NBCe1-dependent HCO 3 -exit from proximal cells and increases pH i . [START_REF] Wang | Fused cells of frog proximal tubule: II. Voltagedependent intracellular pH[END_REF][START_REF] Soleimani | Stoichiometry of Na+-HCO-3 cotransport in basolateral membrane vesicles isolated from rabbit renal cortex[END_REF][START_REF] Sasaki | Mechanism of bicarbonate exit across basolateral membrane of the rabbit proximal convoluted tubule[END_REF] The depolarized proximal cell membrane of mice with deletion of Kcnj15, lower threshold for HCO 3 -, and increased pH i are all consistent with a reduced NBCe1 activity by a voltage-dependent effect. Nevertheless, the involvement of an additional, possibly depolarization-activated, pH i regulatory mechanism cannot be excluded and additional studies will be required to differentiate between these mechanisms.

To date, only the Task2 channel was proposed to sustain NBCe1 activity in proximal tubular cells. [START_REF] Warth | Proximal renal tubular acidosis in TASK2 K+ channeldeficient mice reveals a mechanism for stabilizing bicarbonate transport[END_REF] Although urinary ammonia excretion was not evaluated in Kcnk5(Task2)-deleted mice, the present study indicates that proximal HCO 3 -reabsorption and ammoniagenesis may be regulated by multiple potassium channels types. At present, the respective contributions and concerted actions of Kir4.2 and Task2 remain to be determined because the loss of function of each of them leads to isolated pRTAs of similar severity. This notwithstanding, the phenotype of mice with deletion of Kcnj15, mild compared with that in mice with homozygous NBCe1 deletion, [START_REF] Handlogten | NBCe1 expression is required for normal renal ammonia metabolism[END_REF] was not attenuated by the over-expression of Task2. Actually, the acidosis in mice lacking Kir4.2 is comparable to the severity of the acid-base disturbance caused by haploinsufficiency in Slc4a4, [START_REF] Handlogten | NBCe1 expression is required for normal renal ammonia metabolism[END_REF][START_REF] Gawenis | Colonic anion secretory defects and metabolic acidosis in mice lacking the NBC1 Na+/HCO3-cotransporter[END_REF] and appears consistent with reports showing that a decreased transport by NBCe1 is sufficient to cause pRTA. [START_REF] Horita | Functional analysis of NBC1 mutants associated with proximal renal tubular acidosis and ocular abnormalities[END_REF] In summary (Figure 9), Kir4.2 is a major component of the basolateral barium-sensitive potassium conductance in proximal cells and a new molecular entity through which the kidneys regulate proximal ammonia metabolism, with its loss of function causing a membrane depolarization, an increase in pH i , and a reduced ammoniagenesis. The rise in pH i and lowered threshold for HCO 3 -in mice with deletion of Kcnj15 strongly indicate a reduced driving force for the NBCe1-mediated Na + -HCO 3 -transport as a result of membrane depolarization.

The renal phenotype of mice invalidated for Kcnj15 recapitulates the clinical features of patients with isolated pRTA. [START_REF] Brenes | Impaired urinary ammonium excretion in patients with isolated proximal renal tubular acidosis[END_REF][START_REF] Igarashi | Unraveling the Molecular Pathogenesis of Isolated Proximal Renal Tubular Acidosis[END_REF][START_REF] Lemann | Acid and mineral balances and bone in familial proximal renal tubular acidosis[END_REF] Thus mice with deletion of Kcnj15 spontaneously develop a pRTA in the absence of a generalized proximal dysfunction, that is, outside a Fanconi syndrome, and do not exhibit the expected increases urinary NH 4 + excretion under basal and acid-loading conditions. This makes our observations relevant to human renal physiology and improves our comprehending of acid-base homeostasis and ammonia metabolism. By supporting KCNJ15 as a candidate for human pRTA, our results also bring new insight to the yet unidentified genetic cause of disorders in patients with mild isolated pRTA and lacking mutation in SLC4A4. [START_REF] Katzir | Familial pure proximal renal tubular acidosis--a clinical and genetic study[END_REF][START_REF] Brenes | Impaired urinary ammonium excretion in patients with isolated proximal renal tubular acidosis[END_REF] In cells of the distal convoluted tubule, the closely related Kir4.1 subunit is currently viewed as part of a basolateral K + sensor translating K + homeostasis to regulation of NaCl reabsorption. [START_REF] Welling | Roles and Regulation of Renal K Channels[END_REF] This study expands our view on the functionally relevant renal Kir channels to the proximal tubule and the emergence of Kir4.2 as K + -to-voltage sensor, possibly intimately linking acid-base balance and ammonia metabolism to K + intake, 44 may be anticipated. Adult mice were fed with pellets of a complete 0.25% Na + , 0.6% K + , 0.73% Ca 2+ SAFE-A04 maintenance diet (Usine d'Alimentation Rationnelle, Augy, France) with free access to tap water until the beginning of the experiment. Kidneys retrievals were performed on mice anesthetized by the intraperitoneal injection of ketamine (0.12 mg/g body weight) and xylazine (6 µg/g body weight).

METHODS

Metabolic studies. Metabolic studies were performed in individual metabolic cages (Tecniplast, Decines Charpieu, France) on adult male mice fed with SAFE-A04 powder chow with free access to deionized water alone or supplemented with either 0.28M NH 4 Cl, 0.1 M or 0.28 M NaHCO 3 -and 0.3M sucrose, when appropriate. The methods used for collections and analyses of urine and blood samples were as previously described, [START_REF] Paulais | Renal phenotype in mice lacking the Kir5.1 (Kcnj16) K+ channel subunit contrasts with that observed in SeSAME/EAST syndrome[END_REF] with the modifications detailed in Supplementary Methods section.

Real-time PCR. Real-time PCR on microdissected glomeruli and tubular segments and quantification were as previously described. [START_REF] Lachheb | Kir4.1/Kir5.1 channel forms the major K+ channel in the basolateral membrane of mouse renal collecting duct principal cells[END_REF][START_REF] Paulais | A Na+-and Cl--activated K+ channel in the thick ascending limb of mouse kidney[END_REF] The procedure used for the extraction of whole kidney mRNAs and real-time PCR are detailed in the Supplementary Methods section. The amount of PCR product in each sample was calculated as the percentage of a RNA standard curve established with serial dilutions of a mouse whole kidney cDNA stock solution, [START_REF] Lachheb | Kir4.1/Kir5.1 channel forms the major K+ channel in the basolateral membrane of mouse renal collecting duct principal cells[END_REF][START_REF] Paulais | A Na+-and Cl--activated K+ channel in the thick ascending limb of mouse kidney[END_REF] then revised according to the amount of PCR product for mRNA of the housekeeping RPL26 gene.

The list of all specific primers for mouse genes is given in Table S3.

Immunohistochemistry. Immunohistochemistry was performed on kidney slices using the procedure detailed in Supplementary Methods section. Labeled slices were then examined using either a LSM710 laser scanning confocal microscope or an Axio Scan.Z1 digital slide scanner (Carl Zeiss, Marly Le Roy, France) and 16 bits-depth images were analyzed offline with the Zeiss Zen software. Intensity profiles (see the Results section) were drawn from the corresponding background-substracted signals, normalized to their maximum value over the measurement distance (20 µm).

Western blotting. Western blots were performed on protein extracts in the conditions detailed in Supplementary Methods section. Indirect protein detection was performed by either chemiluminescence using the Pierce™ ECL Western Blotting Substrate (ThermoFischer Scientific) or a iBright Western blot imager (ThermoFisher Scientific). The protein signal was quantified using the ImageJ freeware (NIH, Bethesda, USA) and routinely normalized on -actin signal.

Measurement of membrane potential. The transmembrane potential difference in cells of microdissected proximal tubules was estimated using the non-invasive cell-attached zero current-clamp configuration as described by others investigators, [START_REF] Mason | The interpretation of current-clamp recordings in the cell-attached patch-clamp configuration[END_REF][START_REF] Perkins | Cell-attached voltage-clamp and current-clamp recording and stimulation techniques in brain slices[END_REF] in the conditions detailed in the Supplementary Methods section. The accuracy of this method increases with the resistance ratio R Seal /R Patch+Cell, [START_REF] Perkins | Cell-attached voltage-clamp and current-clamp recording and stimulation techniques in brain slices[END_REF] and low R Seal values may lead to significant membrane potential underestimation. Nevertheless, membrane potential estimates in a given condition were independent of the 1.1-12 GΩ range of R Seal values obtained in this study (Supplementary Figure S6). Because the prolonged zero current-clamp condition almost systematically altered patch seals resistance and prevented reliable paired V m measurements in the presence of Ba 2+ , a separate series of measurements was performed with the blocker continuously present in the bath solution. NBCe1, Na + -coupled HCO The tubules initially were bathed in the bath solution containing Na + and then exposed to Na + -free (Bath Na + withdrawal) then back to Na + -containing (Bath Na + restoration) bath solutions, under constant (23 mM) bath HCO 3 -content. The luminal solution was the HCO 3 --containing and Na + -free solution all throughout the measurement. pH i is the amplitude of the change in pH i induced by the indicated bath Na + manipulation and k is the rate constant. Individual values for each tubule (#) are reported and the means ± SEM are given in the last line. None of the means for Kcnj15 -/-mice is statiscally different from the corresponding mean in Kcnj15 +/+ mice.

Measurements of intracellular

Mice.

  Animals were handled in full compliance with the french government welfare policy. The scientific project and the experimental procedures were favorably evaluated by the Charles Darwin Ethics Committee for animal experimentation (Permit 03-MP/8569/[0]). Mice were housed in the Centre d'Explorations Fonctionnelles (UMRS1138, Paris, France; Permit B75-06-12) and the confined usage of genetically modified organisms (Class 1) was registered with the french Ministère de l'Education Nationale, de l'Enseignement Supérieur et de la Recherche.

  pH in proximal cells. Intracellular pH of isolated proximal tubules microperfused in vitro was assessed by fluorescence imaging microscopy using 2′,7′-Bis-(2-Carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF, ThermoFisher Scientific), as detailed in the Supplementary Methods section. Rate constants (k) of pH i changes were determined by an iterative least-square curvefitting procedure used to fit the data to a single exponential equation of the form as described by other investigators. 26 Statistical analyses. Results are given as means ± SEM for the indicated number of experiments or as individual values. Statistical significance of difference between means was evaluated by bilateral Student's t test using Systat Sigmastat 3.5 software (Ritme Informatique, Paris, France) with significance determined at a P value < 0.05.

Figure 1 .

 1 Figure 1. Mice with deletion of Kcnj15 and Kir4.2 tissular expression. (a) Multiplex

Figure 2 .

 2 Figure 2. Renal Kir4.2 cellular distribution in mice. (a) Kir4.2 mRNA distribution along the mouse nephron. (b) Kcnj15 +/+ (+/+) and Kcnj15 -/-(-/-) mice whole kidneys cross sections probed

Figure 3 .

 3 Figure 3. Subcellular localization of Kir4.2. Fluorescence images (a) and associate intensity profiles (b) of cortical proximal tubular cells of Kcnj15 +/+ mice labeled with 4',6-diamidino-2-

Figure 4 .

 4 Figure 4. Acid-base status and HCO 3 -handling in mice with the deletion of Kcnj15. (a) Basal blood and plasma parameters of Kcnj15 +/+ (+/+, black squares) and Kcnj15 -/-(-/-, red squares) mice. (b) Urine pH and HCO 3 -contents in Kcnj15 +/+ (black squares) and Kcnj15 -/-(red squares) mice as a function of blood HCO 3 -content, under basal condition and after 1 day of the indicated exogenous NaHCO 3 -alkaline loads. Data are from 9 Kcnj15 +/+ and 12 Kcnj15 -/-mice.

Figure 5 .

 5 Figure 5. NH 4 + excretion and distal acidification capabilities in mice with the deletion of Kcnj15. (a) Basal urine parameters of Kcnj15 +/+ (black squares) and Kcnj15 -/-(red squares) mice. (b-d) Blood and urine parameters in 9 to 12 Kcnj15 +/+ (black squares) and 10-11 Kcnj15 -/-(red

Figure 6 .

 6 Figure 6. Expression of mRNA of proteins involved in proximal ammonia metabolism in mice with the deletion of Kcnj15. Renal expressions of mRNAs for the systen N1 Na + and H + -

Figure 7 .

 7 Figure 7. Peptide expression of proteins involved in proximal ammonia metabolism in mice with the deletion of Kcnj15. Renal system N1 Na + and H + -coupled glutamine transporter

Figure 8 .

 8 Figure 8. NBCe1 expression, membrane potential, and intracellular pH of proximal cells. (a) Summary of resting pH i data for 6 Kcnj15 +/+ (+/+, black squares) and 6 Kcnj15 -/-(-/-, red squares) mice. (b) Left panels : expression of the Na + -coupled HCO 3 -cotransporter, isoform 1 (NBCe1) and of the TWIK-related acid-sensitive K + channel 2 (Task2) peptides in 3 Kcnj15 +/+ and 3 Kcnj15 -/-mice. -actin was used as the loading control. Right panel : quantification of NBCE1 and Task2 peptides expression after normalization to their respective expression in Kcnj15 +/+ mice. Data are from 6 Kcnj15 +/+ and 6 Kcnj15 -/-mice. (c) Estimation of membrane potential (V m ) in Kcnj15 +/+ and Kcnj15 -/-mice from cell-attached current-clamp recordings, in the presence (+Ba 2+ ) and absence of 1 mM Ba 2+ in the bath. Left panel : typical V m recordings for Kcnj15 +/+ (black line) and Kcnj15 -/-(red line) mice. At the time indicated by the asterisk, the patch clamp

Figure 9 .

 9 Figure 9. Proposed model for the defective ammonia metabolism in mice with the deletion

Table 1 . Plasma parameters of Kcnj15 +/+ and Kcnj15 -/-mice maintained on a standard diet.

 1 NS, no significant difference between groups.Data are means ± SEM for the numbers of mice given in brackets.

	3	-cotransporter, isoform 1; NHE-3, Na + /H + exchanger 3; PDG,
	phosphate-dependent glutaminase; PEPCK, phosphoenolpyruvate carboxykinase ; SNAT3,
	system N1 Na

+ and H + -coupled glutamine transporter.

Table 2 . Urine parameters of Kcnj15 +/+ and Kcnj15 -/-mice maintained on a standard diet.

 2 BW, body weight; FE, factional excretion; GFR, estimated glomerular filtration rate; NS, no significant difference between groups; Uv, urine volume.Data are means ± SEM for the numbers of mice given in parentheses.

	Bath Na + withdrawal

Table 3 . Kinetics parameters of the changes in pH i of proximal cells induced by bath Na + withdrawal and restoration.

 3

nd : not determined; pH i , intracellular pH.

ACKNOWLEDGMENTS Y.B. and L.P. hold PhD fellowships from the Ministère de l'Enseignement Supérieur et de la Recherche. We thank Nathalie Reveilleau and Stéphanie Baron (Unité d'explorations fonctionnelles métaboliques et rénales in vivo, Hôpital Européen Georges Pompidou, Paris, France) for their expert technical assistance in the measurement of urine NH 4 + and titratable acidity in mice, and Dr Geneviève Escher (Department of Nephrology and Hypertension Inselspital, Universitätsklinik für Nephrologie und Hypertonie, Bern, Switzerland) for aldosterone measurements. We are also grateful to Christophe Klein and Kévin Garbin (Centre d'Imagerie Cellulaire et de Cytométrie, Centre de Recherche des Cordeliers, Paris, France) for their help with microscopy resources, and to the members of the Centre d'Explorations Fonctionnelles and of the Centre de Génotypage et de Biochimie (Centre de Recherche des Cordeliers, Paris, France) for taking care of mice and genotyping.

DISCLOSURE

All the authors declared no competing interests. S1. Physiologic parameters of Kcnj15 +/+ and Kcnj15 -/-mice after an alkaline load. Parameters were measured after a 24-hour oral 0.1 M or 0.28 M NaHCO 3 -induced alkaline load. Results are from 6-18 Kcnj15 +/+ mice and 8-18 Kcnj15 -/-mice. Uv : urine volume. BW : body weight. NS : no significant difference.

SUPPLEMENTARY MATERIAL

RT-qPCR : targeted cDNA (gene) Forward primer Reverse primer Kir4.2 (Kcnj15) 5'-GCAGGCCATAGCAGAG-3' 5'-GTGAGCTTGTATCGCCA-3' NHE-3 (Slc9a3) 5'-TCCCTGGCCTTCATTCG-3' 5'-GTCACCCACCACACACT-3' S3. Primers used for RT-qPCR on whole kidneys and nephron segments extracts and for mice genotyping. Primers used for genotyping were as per Deltagen's instructions. a and b : codons 70-78 and 137-144 of the endogenous Kcnj15 mouse gene, respectively. c is complementary of the 3' sequence of the inserted Neomycin-resistance (LacZ-Neo) gene.

Supplementary methods.

Generation of Kcnj15 -/-mice. SPF breeding nucleus B6;129P2-Kcnf15 tm1Dgen /H strain heterozygous mice of mixed 129P2/OlaHsd and C57BL/6J background (Deltagen, San Mateo, CA, USA) were obtained from the MRC Harwell, Mary Lyon Centre (Oxford, GB). Nucleus mice contained a mutant allele generated by the intragenic deletion of 136 bases (267-402) in exon 4 of the mus musculus Kcnj15 DNA sequence, encoding a 46 amino-acids region ranging from the end of the first membrane-spanning hydrophobic domain (M1) to the pore-forming (H5) domain of the predicted Kir4.2 protein, S1 and by the insertion of a promotorless LacZ reporter gene and a neomycin-resistant gene cassette (LacZ-Neo) flanked by 4.5 Kb (5′) and 2.5 Kb (3′) Kcnj15 genomic DNA fragments homology arms (Figure S7A andS7B). The breeding of nucleus Kcnj15 +/-mice with C57Bl/6 mice (Charles River Laboratories, Saint-Germain-sur-l'Arbresle, France) and the backcrossing of their heterozygous offspring onto a pure C57BL/6 background was then done to produce N5 generation of mice.

The intercrossing of Kcnj15 +/-mice resulted in offspring of usual litter sizes with sexratios of Kcnj15 +/+ , Kcnj15 +/-and Kcnj15 -/-mice at age of weaning close to 1:1 (160 males and 149 females) (Figure S7C). Genotype distribution roughly followed an expected mendelian inheritance pattern with ~30 % Kcnj15 +/+ , ~44 % Kcnj15 +/-and ~26 % Kcnj15 -/-mice. Of the male offspring, ~28 % were homozygous, ~44 % heterozygous and ~28 % Kcnj15 +/+ mice, and female offspring distribution was ~23 % homozygous, ~44 % heterozygous and ~32 % Kcnj15 +/+ mice.

Mice genotype was established by multiplex PCR on ethanol-purified genomic DNA from tail biopsies according to Deltagen's instructions. PCR parameters were 94 °C for 3 min, 35 cycles at 94 °C for 10 sec, 60 °C for 30 sec, 72 °C for 1.5 min, and 72 °C for 7 min and the primers (Eurogentec, Angers, France) used are listed in Table S3. PCR products were separated on a 1.4 % agarose gel along a Invitrogen™ 100bp DNA ladder (ThermoFisher Scientific, Villebon sur Yvette, France) and visualized using ethidium bromide. Endogenous Kcnj15 and targeted alleles were identified by amplification of 224-bp and 430-bp fragments, respectively.

Urine and blood analyses. 24-hour urine samples were collected under water-saturated mineral oil for urine NH 4 + concentration and titratable acidity determination by a DL55 titrator and a ST20 sample changer (Mettler Toledo, Viroflay, France) and for HCO 3 -content measurement by a ABL 720 analyzer (Radiometer, Neuilly-Plaisance, France). Venous blood from the retro-orbital plexus of conscious mice was analyzed by a blood gas epoc ® Reader and Host analyzer (Alere S.A.S., Jouy en Josas, France) for determinations of pH, gas contents and haematocrit, and of Na + , HCO 3 -, Cl -, ionized Ca 2+ , and glucose concentrations. At the end of the experimental period, mice were sacrificed by terminal bleeding after the peritoneal injection of ketamine and xylazine (0.12 and 0.006 mg/g body weight, respectively) and blood samples were collected on heparin for measurements of plasma creatinine by a Konelab 20I analyzer (ThermoFisher Scientific) and of K + concentration by a Sherwood 420 flame photometer (Servilab, Le Mans, France). Urinary Na + and K + contents were measured by flame photometry and urinary aldosterone by a ELISA Kit (Uscn Life Science Inc., Wuhan, China). Glomerular filtration rates were estimated using measured urine and plasma creatinine contents and urine volumes.

Real-time PCR on whole kidneys. Whole kidney mRNAs were extracted using the TRI Reagent ® (ThermoFisher Scientific), treated with the RNAse Free DNAse Set and purified using the RNeasy Mini Kit (Qiagen, Courtaboeuf, France). 1 µg of purified mRNA was reversetranscribed using the first strand cDNA synthesis kit for RT-PCR (Roche Diagnostics, Meylan, France). Real-time PCR was then performed with a LIGHTCYCLER ® 480 using LC 480 SYBR Green 1 Master kit (Roche Diagnostics) in a 10 µl reaction volume, by 45 cycles of 20 seconds temperatures steps at 95 °C, 60 °C, and 72 °C, using the primers listed in Table S3.