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Introduction Anion exchanger 1 (AE1) (SLC4A1 gene product) is a membrane protein expressed in both kidney and red blood cells (RBCs): it exchanges extracellular bicarbonate (HCO3–) for intracellular chloride (Cl–) and participates in acid−base homeostasis. AE1 mutations in kidney α-intercalated cells can lead to distal renal tubular acidosis (dRTA). In RBC, AE1 (known as band 3) is also implicated in membrane stability: deletions can cause South Asian ovalocytosis (SAO). Methods We retrospectively collected clinical and biological data from patients harboring dRTA due to a SLC4A1 mutation and analyzed HCO3– and Cl– transports (by stopped-flow spectrophotometry) and expression (by flow cytometry, fluorescence activated cell sorting, and Coomassie blue staining) in RBCs, as well as RBC membrane stability (ektacytometry). Results Fifteen patients were included. All experience nephrolithiasis and/or nephrocalcinosis, 2 had SAO and dRTA (dRTA SAO+), 13 dominant dRTA (dRTA SAO−). The latter did not exert specific RBC membrane anomalies. Both HCO3– and Cl– transports were lower in patients with dRTA SAO+ than in those with dRTA SAO− or controls. Using 3 different extracellular probes, we report a decreased expression (by 52%, P < 0.05) in dRTA SAO+ patients by fluorescence activated cell sorting, whereas total amount of protein was not affected. Conclusion Band 3 transport function and expression in RBCs from dRTA SAO− patients is normal. However, in SAO RBCs, impaired conformation of AE1/band 3 corresponds to an impaired function. Thus, the driver of acid−base defect during dominant dRTA is probably an impaired membrane expression., Graphical abstract

Pseudohypoaldosteronism type II (PHAII) is a genetic disease characterized by association of hyperkalemia, hyperchloremic metabolic acidosis, hypertension, low renin, and high sensitivity to thiazide diuretics. It is caused by mutations in the WNK1, WNK4, KLHL3 or CUL3 gene. There is strong evidence that excessive sodium chloride reabsorption by the sodium chloride cotransporter NCC in the distal convoluted tubule is involved. WNK4 is expressed not only in distal convoluted tubule cells but also in β-intercalated cells of the cortical collecting duct. These latter cells exchange intracellular bicarbonate for external chloride through pendrin, and therefore, account for renal base excretion. However, these cells can also mediate thiazide-sensitive sodium chloride absorption when the pendrin-dependent apical chloride influx is coupled to apical sodium influx by the sodium-driven chloride/bicarbonate exchanger. Here we determine whether this system is involved in the pathogenesis of PHAII. Renal pendrin activity was markedly increased in a mouse model carrying a WNK4 missense mutation (Q562E) previously identified in patients with PHAII. The upregulation of pendrin led to an increase in thiazide-sensitive sodium chloride absorption by the cortical collecting duct, and it caused metabolic acidosis. The function of apical potassium channels was altered in this model, and hyperkalemia was fully corrected by pendrin genetic ablation. Thus, we demonstrate an important contribution of pendrin in renal regulation of sodium chloride, potassium and acid-base homeostasis and in the pathophysiology of PHAII. Furthermore, we identify renal distal bicarbonate secretion as a novel mechanism of renal tubular acidosis. Pseudohypoaldosteronism type II (PHAII) is a genetic disease characterized by association of hyperkalemia, hyperchloremic metabolic acidosis, hypertension, low renin, and high sensitivity to thiazide diuretics. It is caused by mutations in the WNK1, WNK4, KLHL3 or CUL3 gene. There is strong evidence that excessive sodium chloride reabsorption by the sodium chloride cotransporter NCC in the distal convoluted tubule is involved. WNK4 is expressed not only in distal convoluted tubule cells but also in beta-intercalated cells of the cortical collecting duct. These latter cells exchange intracellular bicarbonate for external chloride through pendrin, and therefore, account for renal base excretion. However, these cells can also mediate thiazide-sensitive sodium chloride absorption when the pendrin-dependent apical chloride influx is coupled to apical sodium influx by the sodium-driven chloride/bicarbonate exchanger. Here we determine whether this system is involved in the pathogenesis of PHAII. Renal pendrin activity was markedly increased in a mouse model carrying a WNK4 missense mutation (Q562E) previously identified in patients with PHAII. The upregulation of pendrin led to an increase in thiazide-sensitive sodium chloride absorption by the cortical collecting duct, and it caused metabolic acidosis. The function of apical potassium channels was altered in this model, and hyperkalemia was fully corrected by pendrin genetic ablation. Thus, we demonstrate an important contribution of pendrin in renal regulation of sodium chloride, potassium and acid-base homeostasis and in the pathophysiology of PHAII. Furthermore, we identify renal distal bicarbonate secretion as a novel mechanism of renal tubular acidosis.

International audience; Distal nephron acid secretion is mediated by highly specialized type A intercalated cells (A-ICs), which contain vacuolar H +-ATPase (V-type ATPase)-rich vesicles that fuse with the apical plasma membrane on demand. Intra-cellular bicarbonate generated by luminal H + secretion is removed by the basolateral anion-exchanger AE1. Chronically reduced renal acid excretion in distal renal tubular acidosis (dRTA) may lead to nephrocalcinosis and renal failure. Studies in MDCK monolayers led to the proposal of a dominant-negative trafficking mechanism to explain AE1-associated dominant dRTA. To test this hypothesis in vivo, we generated an Ae1 R607H knockin mouse, which corresponds to the most common dominant dRTA mutation in human AE1, R589H. Compared with wild-type mice, heterozygous and homozygous R607H knockin mice displayed incomplete dRTA characterized by compensatory upregulation of the Na + /HCO 3 2 cotransporter NBCn1. Red blood cell Ae1-mediated anion-exchange activity and surface polypeptide expression did not change. Mutant mice expressed far less Ae1 in A-ICs, but basolateral targeting of the mutant protein was preserved. Notably, mutant mice also exhibited reduced expression of V-type ATPase and compromised targeting of this proton pump to the plasma membrane upon acid challenge. Accumulation of p62-and ubiquitin-positive material in A-ICs of knockin mice suggested a defect in the degradative pathway, which may explain the observed loss of A-ICs. R607H knockin did not affect type B intercalated cells. We propose that reduced basolateral anion-exchange activity in A-ICs inhibits trafficking and regulation of V-type ATPase, compromising luminal H + secretion and possibly lysosomal acidification.

International audience; Chloride transport by the renal tubule is critical for blood pressure (BP), acid-base, and potassium homeostasis. Chloride uptake from the urinary fluid is mediated by various apical transporters, whereas basolateral chloride exit is thought to be mediated by ClC-Ka/K1 and ClC-Kb/K2, two chloride channels from the ClC family, or by KCl cotransporters from the SLC12 gene family. Nevertheless, the localization and role of ClC-K channels is not fully resolved. Because inactivating mutations in ClC-Kb/K2 cause Bartter syndrome, a disease that mimics the effects of the loop diuretic furosemide, ClC-Kb/K2 is assumed to have a critical role in salt handling by the thick ascending limb. To dissect the role of this channel in detail, we generated a mouse model with a targeted disruption of the murine ortholog ClC-K2. Mutant mice developed a Bartter syndrome phenotype, characterized by renal salt loss, marked hypokalemia, and metabolic alkalosis. Patch-clamp analysis of tubules isolated from knockout (KO) mice suggested that ClC-K2 is the main basolateral chloride channel in the thick ascending limb and in the aldosterone-sensitive distal nephron. Accordingly, ClC-K2 KO mice did not exhibit the natriuretic response to furosemide and exhibited a severely blunted response to thiazide. We conclude that ClC-Kb/K2 is critical for salt absorption not only by the thick ascending limb, but also by the distal convoluted tubule.

International audience; We recently described a novel thiazide-sensitive electroneutral NaCl transport mechanism resulting from the parallel operation of the Cl-/HCO3- exchanger pendrin and the Na+-driven Cl-/2HCO3- exchanger (NDCBE) in β-intercalated cells of the collecting duct. Although a role for pendrin in maintaining Na+ balance, intravascular volume, and BP is well supported, there is no in vivo evidence for the role of NDCBE in maintaining Na+ balance. Here, we show that deletion of NDCBE in mice caused only subtle perturbations of Na+ homeostasis and provide evidence that the Na+/Cl- cotransporter (NCC) compensated for the inactivation of NDCBE. To unmask the role of NDCBE, we generated Ndcbe/Ncc double-knockout (dKO) mice. On a normal salt diet, dKO and single-knockout mice exhibited similar activation of the renin-angiotensin-aldosterone system, whereas only dKO mice displayed a lower blood K+ concentration. Furthermore, dKO mice displayed upregulation of the epithelial sodium channel (ENaC) and the Ca2+-activated K+ channel BKCa. During NaCl depletion, only dKO mice developed marked intravascular volume contraction, despite dramatically increased renin activity. Notably, the increase in aldosterone levels expected on NaCl depletion was attenuated in dKO mice, and single-knockout and dKO mice had similar blood K+ concentrations under this condition. In conclusion, NDCBE is necessary for maintaining sodium balance and intravascular volume during salt depletion or NCC inactivation in mice. Furthermore, NDCBE has an important role in the prevention of hypokalemia. Because NCC and NDCBE are both thiazide targets, the combined inhibition of NCC and the NDCBE/pendrin system may explain thiazide-induced hypokalemia in some patients.

The human ClC-Kb channel plays a key role in exporting chloride ions from the cytosol and is known to be involved in Bartter syndrome type 3 when its permeation capacity is decreased. The ClC-Kb channel has been recently proposed as a potential therapeutic target to treat hypertension. In order to gain new insights into the sequence-structure-function relationships of this channel, to investigate possible impacts of amino-acid substitutions, and to design novel inhibitors, we first built a structural model of the human ClC-Kb channel using comparative modeling strategies. We combined in silico and in vitro techniques to analyze amino acids involved in the chloride ion pathway as well as to rationalize the possible role of several clinically observed mutations leading to the Bartter syndrome type 3. Virtual screening and drug repositioning computations were then carried out. We identified six novel molecules, including 2 approved drugs, diflusinal and loperamide, with Kd values in the low micromolar range, that block the human ClC-Kb channel and that could be used as starting point to design novel chemical probes for this potential therapeutic target.

International audience; Tight regulation of calcium levels is required for many critical biological functions. The Ca2+-sensing receptor (CaSR) expressed by parathyroid cells controls blood calcium concentration by regulating parathyroid hormone (PTH) secretion. However, CaSR is also expressed in other organs, such as the kidney, but the importance of extraparathyroid CaSR in calcium metabolism remains unknown. Here, we investigated the role of extraparathyroid CaSR using thyroparathyroidectomized, PTH-supplemented rats. Chronic inhibition of CaSR selectively increased renal tubular calcium absorption and blood calcium concentration independent of PTH secretion change and without altering intestinal calcium absorption. CaSR inhibition increased blood calcium concentration in animals pretreated with a bisphosphonate, indicating that the increase did not result from release of bone calcium. Kidney CaSR was expressed primarily in the thick ascending limb of the loop of Henle (TAL). As measured by in vitro microperfusion of cortical TAL, CaSR inhibitors increased calcium reabsorption and paracellular pathway permeability but did not change NaCl reabsorption. We conclude that CaSR is a direct determinant of blood calcium concentration, independent of PTH, and modulates renal tubular calcium transport in the TAL via the permeability of the paracellular pathway. These findings suggest that CaSR inhibitors may provide a new specific treatment for disorders related to impaired PTH secretion, such as primary hypoparathyroidism.

The heteromeric inwardly rectifying Kir4.1/Kir5.1 K + channel underlies the basolateral K + conductance in the distal nephron and is extremely sensitive to inhibition by intracellular pH. The functional importance of Kir4.1/Kir5.1 in renal ion transport has recently been highlighted by mutations in the human Kir4.1 gene ( KCNJ10 ) that result in seizures, sensorineural deafness, ataxia, mental retardation, and electrolyte imbalance (SeSAME)/epilepsy, ataxia, sensorineural deafness, and renal tubulopathy (EAST) syndrome, a complex disorder that includes salt wasting and hypokalemic alkalosis. Here, we investigated the role of the Kir5.1 subunit in mice with a targeted disruption of the Kir5.1 gene ( Kcnj16 ). The Kir5.1 −/− mice displayed hypokalemic, hyperchloremic metabolic acidosis with hypercalciuria. The short-term responses to hydrochlorothiazide, an inhibitor of ion transport in the distal convoluted tubule (DCT), were also exaggerated, indicating excessive renal Na + absorption in this segment. Furthermore, chronic treatment with hydrochlorothiazide normalized urinary excretion of Na + and Ca 2+ , and abolished acidosis in Kir5.1 −/− mice. Finally, in contrast to WT mice, electrophysiological recording of K + channels in the DCT basolateral membrane of Kir5.1 −/− mice revealed that, even though Kir5.1 is absent, there is an increased K + conductance caused by the decreased pH sensitivity of the remaining homomeric Kir4.1 channels. In conclusion, disruption of Kcnj16 induces a severe renal phenotype that, apart from hypokalemia, is the opposite of the phenotype seen in SeSAME/EAST syndrome. These results highlight the important role that Kir5.1 plays as a pH-sensitive regulator of salt transport in the DCT, and the implication of these results for the correct genetic diagnosis of renal tubulopathies is discussed.

Tissue kallikrein (TK) is a serine protease synthetized in renal tubular cells located upstream from the collecting duct where renal potassium balance is regulated. Because secretion of TK is promoted by K + intake, we hypothesized that this enzyme might regulate plasma K + concentration ([K + ]). We showed in wild-type mice that renal K + and TK excretion increase in parallel after a single meal, representing an acute K + load, whereas aldosterone secretion is not modified. Using aldosterone synthase-deficient mice, we confirmed that the control of TK secretion is aldosterone-independent. Mice with TK gene disruption ( TK −/− ) were used to assess the impact of the enzyme on plasma [K + ]. A single large feeding did not lead to any significant change in plasma [K + ] in TK +/+ , whereas TK −/− mice became hyperkalemic. We next examined the impact of TK disruption on K + transport in isolated cortical collecting ducts (CCDs) microperfused in vitro. We found that CCDs isolated from TK −/− mice exhibit net transepithelial K + absorption because of abnormal activation of the colonic H + ,K + -ATPase in the intercalated cells. Finally, in CCDs isolated from TK −/− mice and microperfused in vitro, the addition of TK to the perfusate but not to the peritubular bath caused a 70% inhibition of H + ,K + -ATPase activity. In conclusion, we have identified the serine protease TK as a unique kalliuretic factor that protects against hyperkalemia after a dietary K + load.

International audience; alpha- and beta-spectrins are components of molecular scaffolds located under the lipid bilayer and named membrane skeletons. Disruption of these scaffolds through mutations in spectrins demonstrated that they are involved in the membrane localization or the maintenance of proteins associated with them. The ubiquitous alphaII-spectrin chain bears in its central region a unique domain that is sensitive to several proteases such as calpains or caspases. The conservation of this region in vertebrates suggests that the proteolysis of alphaII-spectrin by these enzymes could be involved in important functions. To assess the role of alphaII-spectrin cleavage in vivo, we generated a murine model in which the exons encoding the region defining this cleavage sensitivity were disrupted by gene targeting. Surprisingly, homozygous mice expressing this mutant alphaII-spectrin appeared healthy, bred normally, and had no histological anomaly. Remarkably, the mutant alphaII-spectrin assembles correctly into the membrane skeleton, thus challenging the notion that this region is required for the stable biogenesis of the membrane skeleton in nonerythroid cells. Our finding also argues against a critical role of this particular alphaII-spectrin cleavage in either major cellular functions or in normal development.

International audience; Two nonerythroid homologs of the blood group Rh proteins, RhCG and RhBG, which share homologies with specific ammonia transporters in primitive organisms and plants, could represent members of a new family of proteins involved in ammonia transport in the mammalian kidney. Consistent with this hypothesis, the expression of RhCG was recently reported at the apical pole of all connecting tubule (CNT) cells as well as in intercalated cells of collecting duct (CD). To assess the localization along the nephron of RhBG, polyclonal antibodies against the Rh type B glycoprotein were generated. In immunoblot experiments, a specific polypeptide of Mr approximately 50 kD was detected in rat kidney cortex and in outer and inner medulla membrane fractions. Immunocytochemical studies revealed RhBG expression in distal nephron segments within the cortical labyrinth, medullary rays, and outer and inner medulla. RhBG expression was restricted to the basolateral membrane of epithelial cells. The same localization was observed in rat and mouse kidney. RT-PCR analysis on microdissected rat nephron segments confirmed that RhBG mRNAs were chiefly expressed in CNT and cortical and outer medullary CD. Double immunostaining with RhCG demonstrated that RhBG and RhCG were coexpressed in the same cells, but with a basolateral and apical localization, respectively. In conclusion, RhBG and RhCG are present in a major site of ammonia secretion in the kidney, i.e., the CNT and CD, in agreement with their putative role in ammonium transport.

International audience; Two non-erythroid members of the erythrocyte Rhesus (Rh) protein family, RhBG and RhCG, have been recently cloned in the kidney. These proteins share homologies with specific NH(3)/NH(4)(+) transporters (Mep/Amt) in primitive organisms and plants. When expressed in a Mep-deficient yeast, RhCG can function as a bidirectional NH(3)/NH(4)(+) transporter. The aim of this study was to determine the intrarenal and intracellular location of RhCG in rat kidney. RT-PCR on microdissected rat nephron segments demonstrated expression of mRNAs encoding RhCG in distal convoluted tubules, connecting ducts, and cortical and outer medullary collecting ducts but not in proximal tubules and thick ascending limbs of Henle's loop. Immunolocalization studies performed on rat kidney sections with rabbit anti-human RhCG 31 to 48 antibody showed labeling of the apical pole of tubular cells within the cortex, the outer medulla, and the upper portion of the inner medulla. All cells within connecting tubules had identical apical staining. In cortical collecting ducts, a subpopulation of cells that has either apical staining (alpha-intercalated cells) or diffuse staining (beta-intercalated cells) for the beta1 subunit of the H(+)-ATPase, was heavily stained at their apical pole with the RhCG antibody while principal cells identified as H(+)-ATPase negative cells showed a faint apical staining for RhCG that was much less intense than in adjacent intercalated cells. In the outer medulla and the upper portion of the inner medulla, RhCG labeling was restricted to a subpopulation of cells within the collecting duct that apically express the beta1 subunit of the H(+)-ATPase, indicating that RhCG expression in medullary collecting ducts is restricted to intercalated cells. No labeling was seen in glomeruli, proximal tubules, and limbs of Henle's loop. Immunoblotting of apical membrane fractions from rat kidney cortex with the rabbit anti-human RhCG 31 to 48 antibody revealed a doublet band at approximatively 65 kD.

Calcineurin inhibitors (CNI) are immunosuppressive treatments widely used in transplantation. In mice, CNI activate the renal NaCl cotransporter (NCC), and thereby they cause high blood pressure, metabolic acidosis and hyperkalemia. This association of symptoms is also observed in patients suffering Gordon syndrome, a genetic disease also caused by inappropriate stimulation of NCC. Recently, it has been reported that acidosis of Gordon syndrome also involves stimulation of the Cl-/HCO3- exchanger pendrin. In humans, little is known about the prevalence of Gordon-like syndrome phenotype in patients treated with CNI. Interestingly, NCC and pendrin expression can be assessed by immunoblots using urinary exosomes, small extracellular vesicles that reflect the composition of the plasma membrane of their parental cells. We designed two studies including incident CNI-treated kidney transplants 3 months after transplantation: a retrospective study was designed to determine the prevalence of Gordon-like phenotype, then a prospective one to evaluate NCC and pendrin expression in urinary exosomes. 172 patients were included in the retrospective study: 26% exhibited Gordon-like syndrome phenotype. Then, 58 patients were prospectively included and compared to 58 healthy volunteers. Again ~ 24% of kidney transplant recipients exhibited the Gordon-like phenotype. We show, however, that KT with Gordon-like phenotype do not exhibit a significant increase in NCC, pNCC or pendrin abundance. Furthermore, we show that CNI-treated KT have a marked down regulation of NCC, pNCC protein abundance in urinary exosomes compared to healthy volunteers., Les anticalcineurines sont les immunosuppresseurs les plus utilisés en transplantation rénale. Chez la souris, ils activent le co-transporteur rénal sodium chlore NCC, entrainant un phénotype associant hypertension, acidose métabolique et hyperkaliémie. Ce phénotype est également observé dans une maladie génétique appelée syndrome de Gordon, causée par une stimulation inappropriée de NCC. Récemment, il a été montré que l’acidose dans le Gordon impliquait une stimulation de l’échangeur Cl-/HCO3- pendrine. Chez l’homme, il existe peu de données sur la prévalence d’un phénotype Gordon-like syndrome chez les patients traités par anticalcineurines. De manière intéressante, l’expression de NCC et/ou de pendrine peut être appréhendée par immunoblot en utilisant les exosomes urinaires, qui sont des vésicules extracellulaires dont le contenu dépend de l’activité des cellules émettrices. Nous avons mené deux études qui ont inclus des patients greffés rénaux incidents trois mois après la greffe : Une étude rétrospective pour déterminer la prévalence du phénotype Gordon-like, puis une prospective pour évaluer l’expression de NCC et de pendrine dans les exosomes urinaires. 172 patients ont été inclus dans l’étude rétrospective, dont 26 % présentait le phénotype. Nous avons ensuite recruté 58 patients et 58 volontaires sains. A nouveau, 24 % des greffés présentait le phénotype Gordon-like. Nous montrons, cependant, que les greffés ayant ce phénotype ne présentent pas d’augmentation d’abondance de NCC, pNCC ou pendrine. De plus, nous montrons que les patients greffés sous anticalcineurines ont une abondance significativement moindre de NCC, pNCC dans les exosomes urinaires comparativement aux volontaires.

Kidney plays a major role in several biological fonctions as sodium balance or acid-base homeostasis. Chloride transport in the distal nephron is a key element of these two processes. During my thesis, i have worked on two projects related to physiology and pathophysiology of chloride transport in distal nephron. The first study is the functionnal and molecular characterization of a mouse model bearing the most common dominant dRTA mutation in human AE1, R589H, which corresponds to R607H in the mouse. R607H knock-in mice display incomplete dRTA. Our results showed that reduced basolateral anion exchange activity in type A intercalated cells inhibits trafficking and regulation of V-type ATPase, compromising luminal H+ secretion. In the second study, we investigated the role of a phosphorylation site in the regulation of chloride transport by the ClC-Kb/Barttin channels complex. Our results indicates that Barttin phosphorylation stimulates chloride transport by ClC-Kb channels by increasing the number of active channels in the membrane and hence are involved in the mechanisms of adaptation of renal chloride absorption in response to changes in dietary NaCl intake., Le rein joue un rôle crucial dans de multiples processus biologiques, tels que le maintien de l’homéostasie acide-base et de la balance sodée. Le transport de chlore dans le néphron distal est un élément majeur de ces deux fonctions physiologiques. Au cours de ma thèse, j’ai travaillé sur deux projets relatifs à la physiologie et la physiopathologie du transport de chlore dans le néphron distal. Le premier travail concerne la caractérisation fonctionnelle et moléculaire d’un modèle murin dont le gène Slc4a1 codant pour l’échangeur d’anion de type 1 (AE1) a été modifié, pour introduire une mutation ponctuelle (R589H) dans la séquence protéique. Cette mutation est la plus fréquente des mutations de cette protéine responsable d’acidose tubulaire rénale distale chez l’Homme. Comme les patients, ces souris présentent une acidose tubulaire distale. Nos résultats ont montré que la diminution de l’activité d’échange Cl-/HCO3-, due à la baisse drastique de l’expression d’AE1 dans les cellules -intercalaires du canal collecteur, affecte l’expression et la distribution apicale de la pompe à protons, ce qui altère de ce fait la fonction d’acidification de l’urine. Le second travail porte sur l’étude de la régulation du transport de chlore par le complexe protéique ClC-Kb/Barttin, par phosphorylation de la Barttin. Ce travail démontre que la phosphorylation de la Barttin stimule le transport de chlore du canal ClC-Kb, en augmentant la distribution et la stabilité du complexe ClC-Kb/Barttin à la membrane. Ainsi, la phosphorylation de la Barttin pourrait représenter un mécanisme d’adaptation du transport de chlore en réponse à des variations des apports alimentaires en NaCl.

Kidney plays a major role in several biological fonctions as sodium balance or acid-base homeostasis. Chloride transport in the distal nephron is a key element of these two processes. During my thesis, i have worked on two projects related to physiology and pathophysiology of chloride transport in distal nephron. The first study is the functionnal and molecular characterization of a mouse model bearing the most common dominant dRTA mutation in human AE1, R589H, which corresponds to R607H in the mouse. R607H knock-in mice display incomplete dRTA. Our results showed that reduced basolateral anion exchange activity in type A intercalated cells inhibits trafficking and regulation of V-type ATPase, compromising luminal H+ secretion. In the second study, we investigated the role of a phosphorylation site in the regulation of chloride transport by the ClC-Kb/Barttin channels complex. Our results indicates that Barttin phosphorylation stimulates chloride transport by ClC-Kb channels by increasing the number of active channels in the membrane and hence are involved in the mechanisms of adaptation of renal chloride absorption in response to changes in dietary NaCl intake., Le rein joue un rôle crucial dans de multiples processus biologiques, tels que le maintien de l’homéostasie acide-base et de la balance sodée. Le transport de chlore dans le néphron distal est un élément majeur de ces deux fonctions physiologiques. Au cours de ma thèse, j’ai travaillé sur deux projets relatifs à la physiologie et la physiopathologie du transport de chlore dans le néphron distal. Le premier travail concerne la caractérisation fonctionnelle et moléculaire d’un modèle murin dont le gène Slc4a1 codant pour l’échangeur d’anion de type 1 (AE1) a été modifié, pour introduire une mutation ponctuelle (R589H) dans la séquence protéique. Cette mutation est la plus fréquente des mutations de cette protéine responsable d’acidose tubulaire rénale distale chez l’Homme. Comme les patients, ces souris présentent une acidose tubulaire distale. Nos résultats ont montré que la diminution de l’activité d’échange Cl-/HCO3-, due à la baisse drastique de l’expression d’AE1 dans les cellules -intercalaires du canal collecteur, affecte l’expression et la distribution apicale de la pompe à protons, ce qui altère de ce fait la fonction d’acidification de l’urine. Le second travail porte sur l’étude de la régulation du transport de chlore par le complexe protéique ClC-Kb/Barttin, par phosphorylation de la Barttin. Ce travail démontre que la phosphorylation de la Barttin stimule le transport de chlore du canal ClC-Kb, en augmentant la distribution et la stabilité du complexe ClC-Kb/Barttin à la membrane. Ainsi, la phosphorylation de la Barttin pourrait représenter un mécanisme d’adaptation du transport de chlore en réponse à des variations des apports alimentaires en NaCl.

Hypertension is one of the most common human diseases. Today, many studies support the hypothesis that hypertension necessarily involve abnormal transport of salt (NaCl) by the kidneys. The nephron, the functional unit of the kidney, is composed of several segments with different NaCl transport systems. Our team has recently demonstrated that β-intercalated cells of the collecting duct (CD) were able to reabsorb NaCl. This transport is possible through functional coupling of two exchangers : pendrin and NDCBE. The objective of this thesis is to characterize the transport and show that β-intercalated cells (β-IC) play an important role in maintaining of sodium balance and blood pressure. Through several transgenic mouse models, we have shown that deletion of NDCBE causes hypotension and induces a compensation by another transporter of NaCl : the NCC cotransporter. With another mouse model, knockout for the proton pump H+- ATPase specifically in the intercalated cells and necessary for the reabsorption of NaCI in the β-ICs, we have shown that the β-ICs were capable of modulating the sodium transport of the principal cell of the CD through the secretion of paracrine factors.Otherwise, in mice with a mutated form of the WNK4 kinase, known to cause the Gordon syndrome, a disease characterized by hypertension and hyperkalemia, the NaCl transport by β-ICs is overactive and this overactivation probably contributes to the establishment of the disease., L'hypertension est l'une des maladies humaines les plus courantes. Aujourd'hui, de nombreuses études confirment l'hypothèse selon laquelle l'hypertension implique nécessairement un transport anormal de sel (NaCl) par les reins.Le néphron, l'unité fonctionnelle du rein, est composé de plusieurs segments présentant différents systèmes de transport de NaCl. Notre équipe a récemment démontré que les cellules intercalaires du canal collecteur (CD) étaient capables de réabsorber du NaCl. Ce transport est possible grâce au couplage fonctionnel de deux échangeurs : la pendrine et NDCBE. L'objectif de cette thèse est de caractériser ce transport et de montrer que les cellules intercalaires ( IC) jouent un rôle important dans le maintien de la balance sodée et de la pression artérielle. Grâce à plusieurs modèles de souris transgéniques nous avons pu montrer que la délétion de NDCBE provoque une hypotension et la mise en place d'une compensation par un autre transporteur du néphron : le cotransporteur NCC. A partir d'un autre modèle murin knock-out pour la pompe à proton H+-ATPase spécifiquement dans les cellules intercalaires et nécessaire à la réabsorption de NaCl par les ICs, nous avons montré que les ICs étaient capables de moduler le transport de sodium des cellules principales du CD par la sécrétion de facteurs paracrines. Enfin, chez des souris possédant une mutation de kinase WNK4, connue pour provoquer le syndrôme de Gordon, une maladie caractérisée entre autre par une hypertension artérielle et une hyperkaliémie, le transport de NaCl par les ICs est suractivée et cette suractivation contribue probablement à la mise en place de la maladie.