A Mouse Model of Pseudohypoaldosteronism Type II Reveals a Novel Mechanism of Tenal Tubular Acidosis Irma Karen Lopez-Cayuqueo

A mouse model of pseudohypoaldosteronism type II reveals a novel mechanism of tenal tubular acidosis Irma Karen Lopez-Cayuqueo

To cite this version:

Irma Karen Lopez-Cayuqueo. A mouse model of pseudohypoaldosteronism type II reveals a novel mechanism of tenal tubular acidosis. Tissues and Organs [q-bio.TO]. Sorbonne Université, 2018. English. NNT : 2018SORUS465. tel-02926159

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Sorbonne Université

Ecole doctorale: Physiologie, Physiopathologie et Thérapeutique. ED394

THÈSE DE DOCTORAT

Présentée par: Melle Irma Karen LÓPEZ-CAYUQUEO

A Mouse Model of Pseudohypoaldosteronism type II Reveals a Novel Mechanism of Renal Tubular Acidosis

Dirigée par: Dr. Régine Chambrey

Soutenue le 27-04-2018

Devant un jury composé de:

Prof. Pierre Ronco Président du Jury Prof. Renaud Beauwens Rapporteur Dr. Henrik Dimke Rapporteur Dr. Maria Christina Zennaro Examinateur Dr. Régine Chambrey Examinateur Prof. Jacques Teulon Directeur de thèse

LANGUE DE REDACTION

Avec la permission de l’école doctorale de l’UPMC (ED394), la thèse a été rédigée en anglais, ma langue de recherche et de publication usuelle.

PREFACE

The experiments relative to this work were mostly done during the first 2 years of my PhD under the supervision of Dr. Régine Chambrey at the Paris Cardiovascular Research Center (PARCC).

Part of the experiments shown in this work were performed in collaboration with, or exclusively by professor Pascal Houillier (Figure 17) and Dr. Régine Chambrey (Figure 10). Contribution of all the authors of the paper “A Mouse Model of Pseudohypoaldosteronism type II Reveals a Novel Mechanism of Renal Tubular Acidosis”, submitted to Kidney International, is acknowledged.

During the last year of my PhD, to complete my doctoral formation I performed a long-term internship in the laboratory of Professor Thomas Jentsch at the Leibniz- Forschungsinstitut für Molekulare Pharmakologie im Forschungsverbund (FMP) / Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC). Berlin, Germany. The research performed during this time will not be presented in this thesis.

TABLE OF CONTENTS LIST OF FIGURES ...... 6 LIST OF TABLES ...... 7 LIST OF ABBREVIATIONS ...... 8 ABSTRACT ...... 10 1. INTRODUCTION ...... 11 1.1 Overview of kidney structure and function ...... 11 1.2 Renal physiopathology ...... 13 1.2.1 Sodium handling along the nephron and associated pathologies ...... 14 1.2.2 Potassium handling and its physiological consequences ...... 23 1.3 Acid-base homeostasis ...... 24 1.4 Pseudohypoaldosteronism type II ...... 25 1.4.1 Physiopathology of PHAII ...... 26 1.4.1.1 Regulation of NCC ...... 27 1.4.1.2 Impact of WNK pathway in NCC regulation and in the pathogenesis of PHAII ...... 27 1.4.1.3 Influence of the electroneutral NaCl reabsorption in the pathogenesis of PHAII ...... 33 1.4.1.3.1 Additional information about Pendrin and NDCBE ...... 33 2. AIM OF THE WORK ...... 35 3. MATERIALS AND METHODS ...... 36 3.1 Animal generation and physiological studies ...... 36 3.2 Blood gas and urinary analysis ...... 36 3.3 Potassium diet and ammonium load ...... 37 3.4 Intracellular pH measurements on isolated microperfused tubules ...... 37 3.5 Measure of the fractional volume ...... 38 3.6 Measurements of blood pressure by radiotelemetry ...... 39 3.7 Immunoblotting ...... 39 3.8 Real-time quantitative RT-PCR ...... 40 3.9 Intercalated cell type quantification ...... 41 3.10 Antibody sources ...... 41 3.11 In vitro microperfusion of mouse CCDs and transepithelial ion flux measurement ...... 42 3.12 Statistics and data analysis ...... 43 3.13 Study approval ...... 43 4. RESULTS ...... 44 4.1 TgWnk4PHAII mice exhibit hyperkalemic metabolic acidosis ...... 44 4.2 Electroneutral thiazide-sensitive NaCl transport is activated in the collecting duct of TgWnk4PHAII mice ...... 46 4.3 ENaC-dependent K+ secretion is blocked in TgWnk4PHAII mice despite unchanged renal ENaC activity ...... 48 4.4 Normalization of plasma K+ concentration does not correct metabolic acidosis in TgWnk4PHAII mice ...... 51 4.5 Renal acidosis in TgWnk4PHAII mice is not caused by impaired proton secretion by the distal nephron ...... 52 4.6 Renal acidosis in TgWnk4PHAII mice is a consequence of an increased of the pendrin activity ...... 56

4.7 Hyperchloremic metabolic acidosis and hyperkalemia in TgWnk4PHAII mice are corrected by pendrin genetic ablation...... 60 5. DISCUSSION ...... 65 5.1 The NCC upregulation is not sufficient to explain entire the phenotypic features of PHAII ...... 65 5.2 Pendrin overactivity is contributing to the pathogenesis of PHAII ...... 67 5.3 Impact of pendrin deletion on plasma K+ concentration of TgWnk4PHAII mice ...... 68 5.4 Pendrin hyperactivity as a novel mechanism for renal acidosis ...... 70 5.5 The missing link between pendrin regulation and WNK4 ...... 71 5.6 Conclusion ...... 72 6. PUBLICATIONS...... 74 7. ACKNOWLEDGMENTS ...... 77 8. REFERENCES ...... 78

LIST OF FIGURES

Figure 1. Structure of the nephron……………………………………..……………………….12 Figure 2. Sodium handling along the nephron..……………………………………………….14 Figure 3. The proximal tubule and its main transport processes………….………….……..16 Figure 4. Transports involved in the thick ascending limb of the loop of Henle ...…..…….18 Figure 5. NaCl reabsorption in the distal convoluted tubule..………………………………..19 Figure 6. Main transport processes in the aldosterone-sensitive distal nephron..………...22 Figure 7. Regulation of NCC by WNKs pathway in normal condition and in PHAII..……..32 Figure 8. Protein abundance of NCC and pNCC is increased in TgWnk4PHAII mice ....…..44 Figure 9. TgWnk4PHAII mice exhibit phenotypic abnormalities characteristic of PHAII …...45 Figure 10. Electroneutral NaCl transport system is activated in the CCD of TgWnk4PHAII mice ……………………………………………………………………………………………….47 Figure 11. α-ENaC and the proteolytic cleavage form of γ-ENaC are increased in TgWnk4PHAII mice …………………..…………………………………………………………….49 Figure 12. Identical natriuretic response to amiloride observed in TgWnk4PHAII and control mice ...…………………………………………………………………………………….50

Figure 13. Slight increase in α-BKCa and not changes in ROMK protein abundance in TgWnk4PHAII mice ..…………………………………………………..…………………………..51 Figure 14. Low potassium diet decreases blood plasma K+ concentration in TgWnk4PHAII mice without modifying neither blood pH nor blood [HCO3] …...…….……………….…….52 Figure 15. Metabolic acidosis in TgWnk4PHAII mice is not caused by impaired acid excretion ...…………………………………………………………………………………….....54 Figure 16. Response to an acid load in WNK1PHAII mice indicates a normal acid excretion ...... 55

- - Figure 17. Increased apical Cl /HCO3 exchange activity in β-intercalated cells of TgWnk4PHAII mice ….…………………………………………………………………………….56 Figure 18. The number of β-intercalated cells in TgWnk4PHAII mice is increased ...... 59 Figure 19. Protein abundances of different Na+ transporters in TgWnk4PHAII and TgWnk4PHAII/Pds-/-mice ………………………...... 62 Figure 20. ENaC-dependent K+ secretion is higher after pendrin disruption in TgWnk4PHAII mice ………………...... 63 Figure 21. Double transgenic mice (TgWnk4PHAII/Pds-/-) present not changes in ROMK protein abundance with an increase in α-BKCa abundance ……...... 64

LIST OF TABLES

Table 1. PHAII mutation and their effects on the affected genes ….……………...31 Table 2. Blood parameters of TgWNK4PHAII and TgWNK4PHAII; Pds-/- mice .……..60 Table 3. Blood parameters of TgWNK4PHAII before and 6h after thiazide injection ………………………………………………………………………………….61

LIST OF ABBREVIATIONS

AE1 Anion Exchanger 1 AE4 Anion Exchanger 4 AKI Acute Kidney Injury AQP2 Aquaporin 2 ASDN Aldosterone-Sensitive Distal Nephron BKca Large-conductance Ca2+ -activated K+ Channel CCD Cortical Collecting Duct CD Collecting Duct CKD Chronic Kidney Disease ClCk Chloride Channel k CNT Connecting Tubule DCT Distal Convoluted Tubule ENaC Epithelial Na+ Channel ESRD End-Stage Renal Disease FHHt Familial Hyperkalemic Hypertension HCTZ Hydrochlorothiazide GFR Glomerular Filtration Rate ICs Intercalated Cells IMCD Inner Medulla Collecting Duct KCC3 K+-Cl- Contransporter 3 KCC4 K+-Cl- Contransporter 4

Kir4.1 Inward rectifier-type potassium channel 4.1

Kir5.1 Inward rectifier-type potassium channel 5.1 KS-WNK1 Kidney Specific WNK1 KO Knockout + - NBCe1 Na -HCO3 Cotransporter 1 NCC Na+-Cl- Cotransporter + - - NDCBE Na -driven Cl /HCO3 exchanger

NHE3 Na+-H+ exchanger 3 NKCC2 Na+-K+-Cl- Cotransporter 2 OMCD Outer Medulla Collecting Duct OSR1 Oxidative Stress-Responsive kinase 1

PCs Principal Cells PT Proximal Tubule PHAII Pseudohypoaldosteronism type II Pds Pendrin RTA Renal Tubular Acidosis ROMK Renal Outer Medullary Potassium Channel SGK1 Serum and Glucocorticoid-induced Kinase 1 SPAK STEP20/SPS1-related Proline/Alanine-rich Kinase TAL Thick Ascending Limb TASK2 TWIK Related Acid Sensitive K+ Channel 2 vH+-ATPase Vacuolar-type H+-ATPase WNK With No lysine (K) Kinase WT Wild type

SI units and SI prefixes are used according to the International System of Units. Nomenclature and symbolism for amino acids and peptides follows the guidelines of the IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN): “Nomenclature and symbolism for amino acids and peptides. Recommendations 1983.”

ABSTRACT

Pseudohypoaldosteronism type II (PHAII) is a rare monogenic disease mainly characterized by the association of hyperkalemia, hyperchloremic metabolic acidosis and hypertension. It is produced by mutations in the WNK1, WNK4, KLHL3 or CUL3 genes. The clinical manifestations of PHAII are due, among others, to an increased activity of the thiazide-sensitive Na+-Cl- cotransporter NCC, expressed in the distal convoluted tubule. Several groups attempted to determine how PHAII-causing mutation led to an increased activity of NCC. However, so far the data do not fully explain the physiopathology of PHAII, giving evidence that other renal transport systems are altered in this disease.

It has been reported that WNK4 is expressed not only in the DCT cells but also in -intercalated cells of the cortical collecting duct. These cells exchange

- - intracellular HCO3 for external Cl through pendrin, and therefore, account for renal base excretion. They can also mediate electroneutral thiazide-sensitive NaCl absorption when pendrin-dependent apical Cl- influx is coupled to apical Na+ influx + - - by the Na -driven Cl /HCO3 exchanger NDCBE.

Taking advantage of a mouse model (TgWNK4PHAII) carrying a WNK4 missense mutation (Q562E) identified in PHAII patients, the purpose of this study was to determine whether the electroneutral Na+-Cl- absorption through pendrin/NDCBE is involved in the pathogenesis of this disease.

Our results show that renal pendrin activity is markedly increased in TgWNK4PHAII mice, leading to an increase in thiazide-sensitive NaCl absorption by the collecting duct and contributing to metabolic acidosis. Thus, pendrin genetic ablation in TgWNK4PHAII mice corrects the metabolic acidosis and also the hyperkalemia characteristic of PHAII.

Taken together, our data demonstrate the important contribution of pendrin in renal regulation of NaCl, K+ and acid-base homeostasis and in the pathogenesis of PHAII. Additionally, we identify a renal distal bicarbonate secretion as a novel mechanism of renal tubular acidosis.

1. INTRODUCTION

1.1 Overview of kidney structure and function

The kidneys are involved in a variety of physiological processes including regulation of blood pressure, acid-base homeostasis and volume regulation. The maintenance of a relatively constant extracellular environment is necessary for the cells (and organism) to function normally. This is achieved by excretion of some waste products of metabolism such as urea, creatinine, and uric acid, in addition to water and electrolytes, which are derived primarily from dietary intake. The balance is maintained by keeping the rate of excretion equal to the sum of net intake plus endogenous production.

Anatomically two distinct regions can be identified on the cut surface of a bisected kidney: a pale outer region, the cortex, and a darker inner region, the medulla. In humans the medulla is divided into 8 to 18 renal pyramids. The base of each pyramid is positioned at the cortico-medullary boundary and the apex extends toward the renal pelvis to form a papilla (Brenner and Rector's 2012).

The nephron is the functional unit of the kidney and is composed of the glomerulus located in the Bowman’s capsule and the renal tubules: proximal tubule (PT); the thin limbs and the thick ascending limb of loop of Henle (TAL); distal convoluted tubule (DCT) and connecting tubule (CNT). The last segment of the nephron is the collecting duct (CD); which consists of the cortical collecting duct (CCD), outer medullary collecting duct (OMCD) and the inner medullary collecting duct (IMCD) (Figure 1).

Figure 1. Structure of the nephron. Schematic representation of a longitudinal section of the kidney (left) and representation of a nephron that expands throughout the cortex and medulla (right). The epithelial elements of the nephron include the Bowman’s capsule, the proximal (convoluted and straight) tubule, the thin descending and ascending limbs of the loop of Henle, the thick ascending limb of the loop of Henle, the distal convoluted tubule, the connecting tubule, the initial collecting tubule and the (cortical and medullary) collecting duct. (From Boron and Boulpaep, Medical Physiology, updated 2th edition: 2012).

The cellular constitution and function are different for each renal segment. In general terms, the PT is responsible of the massive reabsorption of water and solutes, around 75%. The thin limbs and TAL are in charge to continue with the reabsorption process but also the setting–up and maintenance of a cortico- papillary gradient of solutes, and hence of osmoles, required for the concentration of final urine. The distal nephron, constituted by DCT, CNT and CD, are responsible of the final reabsorption and secretory processes allowing the organism adjust the final urine according to the body demands.

In summary, in order to maintain a stable milieu intérieur by the selective retention or elimination of water, electrolytes, and other solutes, the kidney is caring out three processes: (1) the filtration of circulating blood from the glomerulus to form an ultra-filtrate of plasma in Bowman’s space; (2) the selective reabsorption across the cells lining the renal tubule (from tubular fluid to blood); and (3) the selective secretion, from peritubular capillary blood to tubular fluid (Boron and Boulpaep 2012).

1.2 Renal physiopathology

Any misbalance in the processes previously described may produce a kidney related pathology. The etiology in many of these diseases has remained puzzling and it is necessary to analyze every case in particular. The three most important clinical problems in nephrology, characterized either by number of patients affected or by rates of associated morbidity and mortality are: end-stage renal disease (ESRD), chronic kidney disease (CKD) and acute kidney injury (AKI) (Brenner and Rector's 2012).

On the other hand, hypertension (high blood pressure) is a major public health problem affecting more than a billion people worldwide with complications, including stroke, heart failure and kidney failure (Trepiccione, Zacchia et al. 2012). According to Arthur Guyton’s hypothesis, the kidney plays a key role in blood pressure control (Guyton, Coleman et al. 1972).

An important aspect in many diseases with renal etiology is the genetic factor. Mutations in some of the major transporters responsible of the reabsorption and excretion of ions along the nephron may produce a diversity of pathologies. The

13 pathophysiological consequences of some of these genetic defects will be discussed in the following part of this thesis.

1.2.1 Sodium handling along the nephron and associated pathologies

Sodium (Na+) being with chloride the principal osmole in the extracellular fluid, renal excretion of salt (NaCl) is the major determinant of the extracellular fluid volume. For this reason, genetic loss or gain of function in renal NaCl transport can be associated with hypotension or hypertension, respectively.

The reabsorption of Na+ takes place along the entire nephron, and very little is excreted in urine (<1% of the filtered load). The proportion of reabsorption differs between nephron segments; thus, the proximal tubule is responsible for the greatest reabsorption (around 67%). The loop of Henle reabsorbs a smaller but significant fraction of filtered Na+ (~25%). And finally, the “fine tuning” of renal NaCl absorption occurs in the distal tubule and collecting duct (~8%) (Figure 2).

Figure 2. Sodium handling along the nephron. Arrows indicate reabsorption of Na+. Numbers indicate the percentage of the filtered load of Na+ reabsorbed or excreted.

The Na+ and Cl− can be reabsorbed through both transcellular and paracellular pathways. In the transcellular pathway, Na+ and Cl− sequentially cross the apical and basolateral membranes before entering the blood. In the paracellular pathway, these ions move entirely along an extracellular route through the tight junctions between cells. In the transcellular pathway, transport rates depend on the electrochemical gradients, ion channels and transporters present at the apical and basolateral membranes. In the paracellular pathway, transepithelial electrochemical driving forces and permeability properties of the tight junctions govern ion movement (Boron and Boulpaep 2012) .

In the proximal tubule, the reabsorption of Na+ can be described as a secondary active transport. Using the Na+ gradient generated by Na+-K+ ATPase pump, the sodium enters the PT cell via a series of transporters that also transport other solutes such as glucose, amino acids, phosphate and citrate. In addition, Na+ entry is coupled to the extrusion of H+ through the electroneutral Na+-H+ exchanger 3 (NHE3), providing at the same time a favorable gradient for the apical Cl− uptake by the Cl--Oxalate/formate exchanger (CFEX) (Edwards 2012). On the basolateral membrane K+ and Cl- are recycled to the blood by the K+-Cl- Cotransporters KCC4 and KCC3, crucial for proximal tubule cell volume regulation (Jentsch 2005). The potassium also is recycled on the basolateral side by K+ channels, such as TASK2, which has been propose as crucial to maintain electrochemical gradients favorable to bicarbonate reabsorption (Lopez-Cayuqueo, Pena-Munzenmayer et al. 2015). In + - the reabsorption of bicarbonate apical NHE3 and basolateral Na -3HCO3 cotransporter (NBC1a) are essential. Finally, it should be noted that, in the proximal tubule, a part of Na+, Cl- and water can be reabsorbed through the paracellular pathway.

Figure 3 represents the main transport processes in the proximal tubule, including the bicarbonate reabsorption, essential for the acid-base homeostasis, which will be further described in the section 1.3.

Figure 3. The proximal tubule and its main transport processes. The proximal tubule is the major reabsorptive segment of the nephron and accounts for reabsorption of nearly 80% of filtered water, sodium, and chloride. In addition, the proximal tubule is the segment where the majority of critical organic solutes such as glucose and amino acids are resorbed. This segment also plays an important role in acid-base balance as it is involved in bicarbonate reabsorption and secretion of organic acids. Transport is powered by Na+-K+ ATPase. NHE3: Na+- + + - - H exchanger 3; NBCe1: Na -HCO3 Cotransporter 1; CFEX: Cl -Oxalate/formate exchanger; AQP1: Aquaporin 1; KCC4/3: K+-Cl- Contransporter 4 and 3; TASK2: TWIK Related Acid Sensitive K+ Channel 2; TJ: Tight Junctions.

In the loop of Henle, around 25% of filtered NaCl is reabsorbed in the thick ascending limb of the loop of Henle where the apical membrane is impermeable to water. This process is essential for the countercurrent mechanism and urine concentration. In the apical membrane of the TAL, sodium and chloride enter the cell via the electroneutral Na+-K+-2Cl- cotransporter (NKCC2), using the favorable inward gradient for sodium generated by the constitutive activity of the basolateral Na+-K+-ATPase pump. A large fraction of the K+ brought into the cell through NKCC2 recycles to the lumen via apical K+ channels, such as ROMK. These channels are essential for replenishing luminal K+ and maintaining adequate Na+- K+-Cl- cotransport. The lumen positivity generated by potassium recycling is able to drive the passive reabsorption of cations (sodium, calcium, and magnesium) between the cells across the tight junction. On the basolateral side, the chloride channel ClC-Kb and its ß-subunit Barttin are essential for Cl- reabsorption (Estevez, Boettgerr et al. 2001), (see below figure 4).

Inactivating mutations in the genes that encode either NKCC2 (Simon, Karet et al. 1996), ROMK (Simon, Karet et al. 1996), ClC-Kb (Simon, Bindra et al. 1997) or Barttin (Birkenhager, Otto et al. 2001) are responsible for the pathogenesis of Bartter’s syndrome. Bartter’s syndrome is a rare inherited disease characterized by the association of severe hypokalemia, metabolic alkalosis, with low or normal blood pressure, high levels of renin and aldosterone in blood, polyuria and dehydration, with hypomagnesemia (Bartter, Pronove et al. 1962).

Figure 4. Transports involved in the thick ascending limb of the loop of Henle. The transport in TAL is characterized by a Na+-2Cl--K+ Cotransporter (NKCC2) on the luminal surface that allows the reabsorption of large amounts of these ions. Reabsorption is powered by a basolateral Na+-K+ ATPase. Importantly, the ascending limb is highly impermeable to water and the reabsorption of large amounts of sodium in the absence of water results in significant dilution of the tubular fluid. A small amount of potassium back-leaks into the lumen via the K+ channel ROMK and yields a positive luminal charge that powers the paracellular reabsorption of positive ions, including magnesium and calcium. The Cl- leaves the cell by the basolateral Cl- channel ClC-Kb. ROMK: Renal Outer Medullary Potassium Channel; NKCC2: Na+-2Cl--K+ Cotransporter; ClC-Kb: Chloride Channel Kb; TJ: Tight Junctions.

In the distal tubule, NaCl reabsorption is almost exclusively transcellular. The NaCl uptake in the apical membrane occurs through the electroneutral Na+-Cl- cotransporter (NCC), highly sensitive to thiazide diuretics. The basolateral step of Na+ reabsorption, as in other cells, is mediated by the Na+-K+ pump and the chloride channel ClC-Kb is also essential in this part of the nephron for the Cl- exit through the basolateral membrane (Hennings, Andrini et al. 2017). The transports in a DCT cell are represented in Figure 5.

The regulation of NCC is crucial for the understanding of related pathologies. Gitelman’s syndrome is an autosomal recessive condition characterized by hypokalemic metabolic alkalosis, hypocalciuria and hypomagnesemia, can be associated with normal or reduced blood pressure (Gitelman, Graham et al. 1966). This syndrome is due to an inactivating mutation of NCC gene (Simon, Nelson- Williams et al. 1996). On the other hand, Gordon syndrome (also called PHAII) is a condition showing opposite symptoms to Gitelman’s syndrome, in which an over activation of NCC is to blame (O'Shaughnessy 2015). Better understanding of the physiopathology of PHAII is one of the aims in the present work and will be discussed in detail in the next pages.

Figure 5. NaCl reabsorption in the distal convoluted tubule. In the distal tubule the filtered sodium load occurs via a luminal Na+-Cl- Cotransporter (NCC) powered by the basolateral Na+-K+ ATPase. NCC is a target of thiazide diuretics. The chloride channel ClC-Kb mediates the basolateral recycling of Cl-. The K+ channels + - Kir4.1 and Kir5.1 mediate the potassium recycling. NCC: Na -Cl Cotransporter; ClC-Kb: Chloride Channel Kb; Kir4.1: Inward rectifier-type potassium channel 4.1; Kir5.1: Inward rectifier-type potassium channel 4.1; TJ: Tight Junctions.

Finally, the aldosterone-sensitive distal nephron (ASDN) is an important site for the final regulation of urinary Na+ excretion. The ASDN is constituted by the late distal convoluted tubule (DCT2), the connecting tubule (CNT), and the collecting duct (CD). The DCT2 is the transition segment between the DCT and the CNT, in which appear, besides DCT cells, cells with the characteristics of the CNT/principal cells (PCs). The CNT and CD are comprised of PCs and several types of intercalated cells (ICs). ICs can be characterized according to the expression of different transporters: (1) α-ICs characterized by apical expression of the V- ATPase and basolateral expression of an anion exchanger AE1, (2) β-ICs with the apical expression of pendrin and V-ATPase in the basolateral side, and (3) non α-, non β-ICs with an apical pendrin and V-ATPase expression (Chambrey and Trepiccione 2015).

The PCs express at the apical membrane the amiloride-sensitive Na+ channel ENaC, formed by 3 homologous subunits: α, β, and γ. ENaC allows the Na+ reabsorption in tandem with the basolateral Na+-K+-ATPase. Consequently, the generation of a negative lumen drives K+ secretion through the apical K+ channel ROMK, allowing PCs to operate as a Na+-K+ exchange. Mutations in the genes encoding ENaC subunits result in rare diseases with either salt loss or retention. Thus, mutations producing gain of function in the β or γ subunit of ENaC are responsible for Liddle’s syndrome, characterized by a severe salt-sensitive hypertension, hypokalemia, and metabolic alkalosis (Warnock 2001). Contrary, loss-of-function mutations in the α, β, or γ subunits of ENaC cause pseudohypoaldosteronism 1B, a severe salt-losing syndrome with hyperkalemia and metabolic acidosis (Scheinman, Guay-Woodford et al. 1999).

Research over the past 20 years has established a paradigm in which PCs are the exclusive site of Na+ absorption while ICs are solely dedicated to acid-base transport. Nevertheless recent studies have revealed the unexpected importance of ICs for NaCl reabsorption. In 2010, Leviel et al. described a novel thiazide- sensitive electroneutral NaCl uptake mechanism in renal ICs, where NaCl is taken - - up from urine by the coordinated action of the Cl -HCO3 exchanger pendrin and + - - Na -driven Cl /2HCO3 exchanger NDCBE (Leviel, Hubner et al. 2010). Later on in 2013, Gueutin et al, showed that while a dysfunction in the β1 subunit of the H+- ATPase in α-IC results in an acidification defect, the dysfunction of the pump in β-

ICs is responsible for renal loss of NaCl, K+, and water (Gueutin, Vallet et al. 2013). Moreover in 2016, Sinning et al, demonstrated that NDCBE is necessary for maintaining sodium balance and intravascular volume during salt depletion or NCC inactivation in mice (Sinning, Radionov et al. 2017). Interestingly, unlike the other absorptive processes in the kidney, β-ICs are rather energized by the H+ V- ATPase than the Na+-K+ pump (Chambrey, Kurth et al. 2013).

A summary of the main transporters involved in sodium handling in CNT and CCD is represented in figure 6.

Figure 6. Main transport processes in the aldosterone-sensitive distal nephron. Principal cells harbor luminal sodium (ENaC) and potassium (ROMK) channels that allow the sodium reabsorption and potassium secretion, respectively. This process is powered by a basolateral Na+-K+ ATPase. Principal cells also display a regulated transcellular permeability to water in response to vasopressin via aquaporin 2 (AQP2) water channels. The α-intercalated cells are characterized by the capacity to secrete protons via a luminal H+-ATPase and to reabsorb potassium through the H+-K+ ATPase. Finally, the NaCl reabsorption in β-intercalated cells is generated by two cycles of pendrin (Pds) coupled with one cycle of NDCBE. This process results in a net uptake of one Na+, one Cl- and two - - HCO3 ions, whereas one Cl ion is recycled across the apical membrane. Then, one Cl- ion exits the cell through the basolateral chloride channel ClC-Kb, while Na+ and bicarbonate exit the cell at the basolateral membrane via AE4. ROMK: Renal Outer Medullary Potassium Channel; ClC-Kb: Chloride Channel Kb; Pds: + - - Pendrin; NDCBE: Na -driven Cl /HCO3 exchanger; AE1: Anion Exchanger 1; AE4: Anion Exchanger 4; TJ: Tight Junctions.

1.2.2 Potassium handling and its physiological consequences

The distribution of potassium (K+) in the body differs strikingly from that of Na+. Whereas Na+ is largely extracellular, K+ is the most abundant intracellular cation. Maintenance of extracellular K+ concentration within a narrow range is vital for numerous cell functions, particularly electrical excitability of cardiac cells and neurons. When plasma K+ concentration deviates from normal, serious adverse consequences, such as cardiac arrhythmias and death, can result (Palmer 2015).

The proximal tubule consistently reabsorbs the bulk of the filtered K+ (~67%), whereas the distal nephron may reabsorb or secrete K+. Depending, among others, on dietary K+ intake, aldosterone and insulin levels, adrenergic activity, osmolality and acid-base status, the K+ excretion can vary widely from 1% to 110% of the filtered load (Costanzo 2011).

K+ reabsorption in the proximal tubule is largely passive and follows the movement of Na+ and water. Along the TAL, K+ reabsorption amounts to around 20% of filtered K+. In part, it is coupled to Na+ absorption and involves the Na+-K+-2Cl- cotransporter (NKCC2) in the luminal membrane but it is also largely paracellular.

In the distal nephron, depending on the body demands, either reabsorption or secretion of K+ can take place. The α-intercalated cells are responsible for K+ absorption, which occurs via an apical K+ uptake mediated by the H+-K+-ATPase, followed by passive efflux of K+ across the basolateral membrane. This process occurs only on a low-K+ diet (K+ depletion), when K+ excretion can be as low as 1% of the filtered load. Potassium secretion occurs in the principal cells, where ENaC, the apically-located Na+ channel plays a critical role by providing intracellular Na+ as substrate for the Na+-K+-ATPase and by depolarizing the apical membrane, thereby increasing the driving force for K+ efflux into the tubular fluid. At least two types of apical K+ channels have been implicated in K+ secretion, ROMK and BK (Welling 2016).

The mechanisms by which aldosterone promotes apparently opposite effects like Na+ reabsorption and K+ secretion, remain poorly understood. Volume depletion leads to a Na+ retaining state, at least in part, by the action of aldosterone. In this condition, while reabsorption of salt is increased, K+ secretion remains unchanged. Thus salt is retained without losing K+. Instead, if plasma K+ is increased,

23 aldosterone is also released, favoring K+ secretion in the distal nephron, without affecting the salt reabsorption rate. Thus K+ is lost without retaining salt. This is commonly referred to as the aldosterone paradox (Arroyo, Ronzaud et al. 2011). In brief, hyperaldosteronism increases K+ secretion and causes hypokalemia. And hypoaldosteronism decreases K+ secretion and causes hyperkalemia.

In addition, acid-base disturbances also have complex effects on renal K+ excretion. A fall in intracellular pH reduces the activity of Na+-K+ ATPase on the basolateral membrane and therefore diminishes activity of ENaC, ROMK and BK on the apical membrane, contributing to an inhibition of active K+ secretion. Furthermore, stimulation of electrogenic H+ secretion by the vacuolar ATPase in intercalated cells during acidemia tend to reduce the lumen-negative, transepithelial potential difference, thus inhibiting K+ secretion. Moreover, acidemia upregulates apical H+-K+ ATPase in intercalated cells, thereby enhancing K+ reabsorption and reducing net K+ secretion. On the contrary, alkalemia stimulates K+ secretion in the distal nephron, by increasing activities of ENaC, ROMK, and BK (Aronson and Giebisch 2011).

From a pharmacological point of view, loop diuretics or thiazide diuretics, increase the driving force for K+ secretion, favoring hypokalemia while K+-sparing diuretics decrease K+ secretion producing hyperkalemia.

1.3 Acid-base homeostasis

Maintaining systemic acid-base balance is critical because the majority of biological processes are dependent on pH. Plasma pH is controlled by the respiratory and urinary systems, which regulate plasma PCO2 and bicarbonate − − (HCO3 ) concentrations, respectively. The association between PCO2 and HCO3 - is given by the Henderson-Hasselbalch equation: pH = pK + log ([HCO3 ]/s ×

PCO2), where “s” is solubility of CO2 at a given temperature and “pK” the − dissociation constant of the CO2/HCO3 buffer system (Boron and Boulpaep 2012).

- − The kidneys play a major role in HCO3 handling by reabsorbing filtered HCO3 and − generating new HCO3 , which is absorbed and used to titrate acid yielded by protein breakdown during metabolic processes. Abnormalities in those processes

24 will eventually lead to a disruption in systemic acid-base balance and provoke metabolic acid-base disorders.

The proximal tubule contributes to renal acid-base regulation by absorbing most of filtered bicarbonate (∼85%), generating new bicarbonate via ammoniagenesis, and secreting ammonia into lumen, which ultimately acts as the major base form of + buffer (as NH3) used to titrate, trap, and excrete proton into urine (as NH4 ). The thick ascending limb contributes to renal acid-base regulation by absorbing majority of the remain filtered bicarbonate (∼10%) and absorbing luminal ammonia to accumulate sufficiently high ammonia concentration in medullary interstitial fluid (Eladari and Kumai 2015).

The distal parts of the nephron also play a critical role in renal acid excretion, and thus, in acid-base homeostasis. Acid secretion is achieved by α-ICs, where the protons generated from the hydration of CO2 within these cells are extruded actively across the apical membrane by the vH+-ATPase pump, while bicarbonate ions, which are also produced by this process, are translocated across the - - basolateral membrane by the Cl /HCO3 exchanger AE1. Dysfunction of either the pump or the anion exchanger can block proton secretion and produce an accumulation of acid in the organism. This defect is named distal renal tubular acidosis (dRTA) type I (Alper 2002). The β-ICs also contribute to acid-base - - balance, by secreting bicarbonate through the Cl -HCO3 exchanger pendrin.

1.4 Pseudohypoaldosteronism type II

Hypertension is one of the most common diseases in the general population. This disorder is a major risk factor for many common causes of morbidity and mortality including stroke, myocardial infarction, congestive heart failure and end-stage renal disease (Lifton, Gharavi et al. 2001). Despite its importance in human pathology, the molecular mechanisms underlying hypertension are still poorly understood in the majority of patients.

However, several rare Mendelian blood pressure syndromes have shed light on the molecular mechanisms involved in deregulated ion transport in the distal kidney. One of them in particular is pseudohypoaldosteronism type II (PHAII). PHAII also known as Familial hyperkalemic hypertension (FHHt) or Gordon

25 syndrome; is an autosomal-dominant disorder characterized by salt-dependent hypertension, hypercalciuria, suppressed plasma renin and variable levels of aldosterone, hyperchloremic metabolic acidosis and hyperkalemia despite normal renal function in terms of an intact glomerular filtration rate (GFR) (Gordon, 1986).

1.4.1 Physiopathology of PHAII

Mutations in two members of the with-no-lysine (K) serine/threonine kinases (WNK) family, WNK1 or WNK4, (Wilson, Disse-Nicodeme et al. 2001) or in their regulators KELCH3 (Louis-Dit-Picard, Barc et al. 2012) and Cul3 (Boyden, Choi et al. 2012), were identified as causing PHAII. All mutations lead to abnormal phosphorylation of the Na+-Cl− Cotransporter (NCC) expressed on DCT, increasing its expression and activity.

The precise mechanism by which PHAII-causing mutations lead to the different phenotypic abnormalities characteristic of this syndrome is not fully elucidated. The excess reabsorption of salt in the DCT increases vascular volume resulting in thiazide-sensitive hypertension. Patients with PHAII respond to aggressive salt- restriction or relatively small doses of thiazide diuretics, suggesting that indeed NCC gain of function is the major cause of the development of the disease. By contrast, the pathogenesis of hyperkalemia and acidosis in PHAII seems to be more complex and is poorly understood.

It has been proposed that the reduced Na+ delivery to the lumen of downstream nephron segments (CNT and CD) reduces ENaC activity, and thereby, prevents the development of a lumen negative transepithelial voltage. Simultaneously, it is proposed that PHAII-causing mutations reduce the activity of ROMK in the apical membrane of PCs. Both effects would act synergistically to block potassium secretion and provoke hyperkalemia. The molecular basis responsible of the metabolic acidosis in PHAII is not clear either. Acidosis might result from the absence of ENaC-dependent transepithelial potential difference or could be due to the reduced ammonia production and secretion following hyperkalemia.

1.4.1.1 Regulation of NCC

Hormones such as vasopressin (Saritas, Borschewski et al. 2013), parathyroid hormone (Ko, Cooke et al. 2011) or insulin (Chavez-Canales, Arroyo et al. 2013) might participate to NCC regulation. However, it is recognized that the renin– angiotensin–aldosterone system (RAAS) is the major regulatory system of NCC.

During NaCl restriction, plasma aldosterone increases, thereby activating both the transcription and protein abundance of ENaC (Masilamani, Kim et al. 1999). This in turn stimulates Na+ absorption by the distal nephron, and Na+ retention. In parallel, aldosterone also stimulates thiazide-sensitive Na+ reabsorption in the DCT, an effect correlated with an increase in NCC abundance (Kim, Masilamani et al. 1998). Furthermore, angiotensin II, which production is stimulated during volume depletion to keep blood pressure constant by favoring renal NaCl retention and vascular vasoconstriction stimulates most of renal Na+ transporters, including NCC (San-Cristobal, Pacheco-Alvarez et al. 2009).

Some studies have established that chronic and acute K+ loading decrease NCC activity (Sorensen, Grossmann et al. 2013). Therefore NCC is inversely regulated in two situations of elevated aldosterone, i.e., increased during NaCl restriction and decreased during K+ loading. The difference between these two situations could derive from the level of circulating AngII. While AngII level is high during NaCl restriction, it is low during K+ load (Eladari, Chambrey et al. 2014). The WNK pathway seems to be the molecular switch coordinating the NCC response to these two physiological states, as we will discuss below.

1.4.1.2 Impact of WNK pathway in NCC regulation and in the pathogenesis of PHAII

The WNK kinases (With No K (lysine)), comprise a serine-threonine kinase subfamily characterized by the lack of a highly conserved lysine residue in subdomain II of the kinase domain, expressing instead a cysteine. Mutations in the genes encoding for two members of the WNK kinase family, WNK1 and WNK4, were shown to cause the human monogenic disease PHAII (Wilson, Disse- Nicodeme et al. 2001). PHAII mutations in the WNK1 gene are large intronic deletions that cause an increase in WNK1 expression while WNK4 mutations are

27 missense mutations clustered in a short acidic segment distal to the kinase domain, also resulting in increased expression of WNK4 due to a decrease in its degradation.

WNK1 and WNK4 together with WNK3 form a complex network involved in the regulation of ion transport in the distal nephron (Murthy, Kurz et al. 2017). WNKs activate NCC by phosphorylating and activating of two homologous kinases, SPAK (STE20 (Sterile 20)/SPS-1 related proline/alanine-rich kinase) and OSR1 (Oxidative Stress-Responsive 1). These, in turn, directly phosphorylate NCC along its amino terminal cytoplasmic domain (Moriguchi, Urushiyama et al. 2005). The phosphorylation events activate the transport protein, leading to increase solute transport, in part by stabilization of NCC in the plasma membrane. However, the role of WNKs pathway in the sodium handling and blood pressure regulation, has been never totally clear because, contradictory results have been obtained with in vitro experiments. The analysis of genetically modified mouse models has partly clarified our knowledge on WNKs.

In particular, within the last few years, it has been proposed that chloride plays an important role in the regulation of WNKs. Changes in osmolality and Cl- concentration activate WNK kinases. When the WNKs are expressed in heterologous systems with NCC, exposure to hypotonic low Cl- medium increases the abundance of phosphorylated NCC and NCC activity because WNKs activate endogenous SPAK or OSR1. Furthermore, when chloride binds the WNK1 catalytic domain, it inhibits its autophosphorylation and kinase activity (Piala, Moon et al. 2014). According to one model, small changes in plasma K+ concentration affect NCC via a secondary alteration in the intracellular Cl- concentration, thereby modulating WNK-SPAK/OSR1-NCC cascade (Terker, Zhang et al. 2016). Thus, the WNK kinase pathway is considered to fine tune the balance between electroneutral NaCl absorption by NCC in the DCT and electrogenic “Na+/K+ exchange” promoted by ENaC in the downstream CNT/CD. This would provide a mean to regulate Na+ balance without disturbing K+ secretion during hypovolemia or increased K+ secretion without impairing Na+ balance when K+ intake is high and vice versa (Kahle, Wilson et al. 2005). It has been shown recently that the Kir4.1/Kir5.1 K+ channel are absolutely required for DCT cells to respond to plasma

K+ concentration changes and maintain normal K+ homeostasis (Cuevas, Su et al. 2017).

WNK4 is expressed in cells from the thick ascending limb to the collecting duct (Ohno, Uchida et al. 2011), where NCC is its principal target. While WNK4 expression in heterologous systems typically inhibits NCC activity (Chavez- Canales, Zhang et al. 2014), WNK4 dominant effect in vivo is to activate NCC (Ohta, Rai et al. 2009). Thus, WNK4 deletion in a mouse model nearly abrogates NCC activity (Castaneda-Bueno, Cervantes-Perez et al. 2012)

Although WNK1 can also phosphorylate and activate SPAK/OSR1 in vitro, its physiological action along the mammalian distal nephron has been more difficult to resolve. WNK1 deletion is embryonic lethal, and complete deletion of WNK1 in kidney has not been reported. In fact, the predominant WNK1 isoform in the distal nephron lacks a kinase domain; it is called kidney-specific WNK1 (KS-WNK1) (Delaloy, Lu et al. 2003). KS-WNK1 cannot phosphorylate substrates and instead interacts with and modulate the action of other WNKs via a carboxyl terminal HQ domain (Chavez-Canales, Zhang et al. 2014). WNK1-associated PHAII, which results from a large deletion within intron 1, leads to ectopic expression of the full- length WNK1 (L-WNK1) isoform in the DCT. Thus introducing a kinase-active WNK1 isoform in a segment that normally exhibits little WNK1 kinase activity (Vidal-Petiot, Elvira-Matelot et al. 2013),

In 2012, the discovery that mutations in two other genes, cullin 3 (CUL3) and kelch-like 3 (KLHL3), also cause PHAII again has broadened our understanding of the WNK kinase network. These proteins target WNKs by ubiquitination, inducing their degradation through the proteasome. KLHL3 is an adaptor protein, which associates WNK with Cullin 3, part of an E3 ligase that mediates ubiquitination. Disease-causing mutations in KLHL3 impair either KLHL3 binding to WNK1/4 or KLHL3 binding to Cullin 3 (Ohta, Schumacher et al. 2013). When WNKs cannot associate with Cullin 3, they are no longer prone to degradation, they accumulate in cells, and enhance NCC activity. Interestingly, disease-causing mutations within the acidic domain of WNK4 also disrupt its ability to bind KLHL3, suggesting that the fundamental mechanisms for disease caused by WNK4 and KLHL3 mutations are similar (Shekarabi, Zhang et al. 2017). On the other hand, the mechanism by which Cullin 3 mutations cause PHAII is not as clear. In a mouse model carrying

29 the PHAII Cullin 3 producing-mutation (deletion of the exon 9), WNK4, WNK1 and NCC abundance are increased (Schumacher, Siew et al. (2015). However, the mutant protein does not appear to exhibit complete loss of function because deletion of Cullin 3 is embryonic lethal (Singer, Gurian-West et al. 1999). Another group has reported that PHAII-causing mutation in Cullin 3, increased KLHL3 ubiquitination and degradation (McCormick, Yang et al. 2014).

The table 1 shows PHAII-causing mutations and theirs effect on the affected genes, and Figure 7 illustrates the effect of PHAII causing mutation in the regulation of NCC.

Table 1. PHAII mutation and their effects on the affected genes

Gene Effect Result Effect on the encoded protein WNK1 Deletion of intron 1 Increased WNK1 L-WNK1 expression

WNK4 Missense mutation in Increased WNK4 WNK4 the acidic motif expression due to disruption in the KLHL3 recognition site

WNK4 R1185C mutation in Disruption of Unknown the C-terminal domain regulatory mechanism involving calmodulin binding and SGK1 phosphorylation sites

KLHL3 Missense mutations in Disruption of the WNK1, WNK4, the BTB or BACK CUL3–KLHL3 WNK3 domain interaction

KLHL3 Missense mutations in Disruption of the WNK1, WNK4, the Kelch propeller substrate (WNK) WNK3 blades binding

CUL3 Exon 9 deletion Increased KLHL3 KLHL3 ubiquitination and WNK1, WNK4, degradation. WNK3 Altered CUL3 flexibility leading to CUL3 auto- degradation and prevention of WNK ubiquitination

Modified from Murthy et al. Cell. Mol. Life Sci. 2017, (Murthy, Kurz et al. 2017)

Figure 7. Regulation of NCC by WNKs pathway in normal condition and in PHAII. Under physiological conditions, protein levels of WNK1 and WNK4 in DCT are maintained by degradation through ubiquitination by the KLHL3–CUL3 E3 ligase complex. However, PHAII-causing mutations in the acidic domain of WNK4 as well as, in the Kelch domains of KLHL3, interrupt binding of the KLHL3–CUL3 E3 ligase complex, resulting in an impaired ubiquitination and degradation of WNK kinases. PHAII-causing CUL3 mutations lack a portion of exon 9 and cause decreased ubiquitination and degradation of WNK kinases. Thus, PHAII-causing mutations in three different genes share a common mechanism: decreased ubiquitination and increased WNK1/WNK4 protein levels in DCT cells. Both WNK1 and WNK4 are increased in the kidneys with PHAII caused by KLHL3 and CUL3 mutations. Ub: ubiquitin; DCT: distal convoluted tubule; NCC: Na+-Cl- Cotransporter. (From Sohara et al. 2016, Nephrology Dialysis Transplantation (Sohara and Uchida 2016)).

1.4.1.3 Influence of the electroneutral NaCl reabsorption in the pathogenesis of PHAII

The WNK4 kinase is expressed in the DCT but interestingly also in β-intercalated cells of the CCD (Ohno, Uchida et al. 2011). These cells are important for renal - - base excretion via pendrin, which exchanges intracellular HCO3 for external Cl . Moreover, β-intercalated cells can also mediate thiazide-sensitive NaCl absorption when pendrin-dependent apical Cl- influx is coupled to apical Na+ influx by the Na+- - - driven Cl /HCO3 exchanger NDCBE.

Overexpression of pendrin in intercalated cells can favour the onset of chloride- dependent hypertension, suggesting a role in the regulation of blood pressure in vivo for this exchanger (Jacques, Picard et al. 2013). Conversely, pendrin-deficient mice are protected against mineralocorticoid-induced hypertension (Verlander, Hassell et al. 2003) and develop a lower blood pressure during NaCl restriction (Wall, Kim et al. 2004). Furthermore, an acute inactivation of pendrin results in a low blood pressure (Trepiccione, Soukaseum et al. 2017). Despite all the evidences about the importance of ICs and pendrin in the NaCl balance, thereby in the control of blood pressure, their impact in the pathophysiology of PHAII has not been studied so far.

1.4.1.3.1 Additional information about Pendrin and NDCBE

The anion exchanger Pendrin (Slc26a4) is expressed in the kidney, thyroid and inner ear. Inactivating mutations of pendrin gene in humans lead to Pendred syndrome, an autosomal recessive disease characterized by sensorineural deafness associated with hypothyroidism and goiter (Scott, Wang et al. 1999). In kidney, pendrin is expressed in CD and CNT as well as in a few cells of the DCT2. Within these segments, pendrin localizes in the apical membrane and in apical cytoplasmic vesicles of both β-IC and non-α- and non-β-ICs (Wall and Lazo- Fernandez 2015).

− − Pendrin-mediated Cl /HCO3 exchange is greatly upregulated in models of metabolic alkalosis, such as following aldosterone administration or dietary

NaHCO3 loading (Wall 2017). It is also upregulated by angiotensin II. Moreover, pendrin modulates aldosterone-induced Na+ absorption by modifying ENaC

33 abundance and function through a kidney-specific mechanism that does not involve changes in the concentration of a circulating hormone. Instead, pendrin − changes ENaC abundance and function at least in part by altering luminal HCO3 and ATP concentrations. Pendrin has also been shown to participate in the release of catecholamines by adrenal glands (Lazo-Fernandez, Aguilera et al. 2015); this is another mechanism by which pendrin can potentially affect blood pressure.

- In summary, although pendrin-mediated HCO3 secretion plays a role in the renal regulation of acid-base balance, pendrin-mediated Cl- absorption is probably more physiologically relevant by its contribution to the regulation of vascular volume and blood pressure.

+ - - NDCBE protein (Na -driven Cl /HCO3 exchanger, encoded by the gene Scl4a8) is expressed in the brain where it modulates the process of synaptic glutamate release (Sinning, Liebmann et al. 2011). In the kidney, functional data have shown that NDCBE localizes to the intercalated cells (Leviel, Hubner et al. 2010), where during dietary NaCl restriction, it mediates electroneutral NaCl absorption in the - - CCD together with pendrin-mediated Cl /HCO3 exchange (Eladari, Chambrey et al. 2012). The localization of NDCBE in the kidney by immunofluorescence has not been determined but its presence has been demonstrated in CCD by immunoblotting of isolated CCD (Eladari and Hubner 2011).

2. AIM OF THE WORK

The pathogenesis of PHAII has remained puzzling for decades. Increased NCC activity per se is not sufficient to explain all the phenotypic characteristics observed in PHA II patients, particularly the metabolic acidosis and hyperkalemia. Since WNK4, one of the genes mutated in PHA II patients is not only expressed in the DCT but also in CCD, where alongside the amiloride-sensitive epithelial Na+ channel ENaC, NaCl reabsorption is performed by the coordinated action of the Cl- - + - - /HCO3 exchanger pendrin/SLC26A4 and the Na -driven Cl /2HCO3 exchanger NDCBE/SLC4A8, we hypothesized that the pendrin/NDCBE system was essential in the pathogenesis of PHAII.

The aim of this study was to investigate whether the pendrin/NDCBE system is upregulated in a mouse model carrying one of the WNK4 missense mutations identified in PHAII patients (WNK4-Q562E; TgWNK4PHAII mice), and in this manner contributes to the development of this disease.

3. MATERIALS AND METHODS

3.1 Animal generation and physiological studies

TgWNK4PHAII mice (Lalioti, Zhang et al. 2006) were provided by Dr. Richard P. Lifton (Yale University, New Haven Connecticut, USA). Pds-/- mice (Everett, Belyantseva et al. 2001) were provided by Dr. Andrew Griffith (NIDCD, NIH, Rockville, Maryland, USA). TgWNK4PHAII animals, carrying two copies of the mutated WNK4 gene, were bred with Pds-/- animals to generate double- heterozygote animals. Those were then bred with TgWNK4PHAII animals in order to obtain Pds+/- animals with two copies of the mutated WNK4 gene. TgWNK4PHAII/Pds+/- were then intercrossed to generate TgWNK4PHAII;Pds+/+ and TgWNK4PHAII;Pds-/-. In parallel, Pds-/- animals were crossed with C57Bl/6J mice over two generations in order to generate Pds+/- animals on the same mixed genetic background than the double-mutant mice, as TgWNK4PHAII animals are from a C57Bl/6J background. The resulting Pds+/- mice were intercrossed to obtain Pds+/+ and Pds-/- mice.

The experimental treatments were conducted on 3-4 months-old, TgWnk4PHAII or TgWnk4PHAII/Pds-/- mice, and their wild-type littermates. The mice were bred and maintained accordingly to French Ministry of Agriculture (Authorization Executive Order A7515320) for scientific experimentation on animals, European Communities Council Directive, and international ethical standards.

3.2 Blood gas and urinary analysis

Blood was sampled from the retro-orbital sinus of anaesthetized mice (isofluorane 2.5%) in order to measure plasma electrolytes and blood pH. The subsequent blood analyses were done on an ABL 77 pH/Blood-Gas Analyzer (Radiometer). Urinary electrolytes were measured with a flame photometer (IL943; Instruments Laboratory, Lexington, MA) in samples diluted 1:4 in distillated water. Urine aldosterone was measured by RIA (DPC Dade Behring, now Siemens Healthcare, Erlangen, Germany). All urinary values were adjusted by urinary creatinine previously quantified by Jaffé colorimetric method.

3.3 Potassium diet and ammonium load

Mice were fed either with normal chow containing 0.32% of Na+, 0.43% of Cl- and 0.81% of K+, or with K+ free diet containing 0% K+, 0.43% of Cl- and 0.32% of Na+ (INRA, Jouy-en-Josas, France). For the acid load treatment, the mice were given free access either to distilled water or water containing 0.28 M NH4Cl for seven days.

3.4 Intracellular pH measurements on isolated microperfused tubules

During intracellular pH measurement experiments, the average tubule length exposed to bath fluid was limited to 300 – 350 µm in order to prevent motion of the tubule. Two solutions were used, differing in their content in chloride. The composition of the solutions were as follows; chloride-containing solution was composed of 119 mM NMDG-Cl, 23 mM NMDG-HCO3, 2 mM K2HPO4, 1.5 mM

CaCl2, 1.2 mM MgSO4, 10 mM HEPES, and 5.5 mM D-glucose; chloride-free solution was composed of 119 mM NMDG-gluconate, 23 mM NMDG-HCO3, 2 mM

K2HPO4, 7.5 mM Ca-gluconate, 1.2 mM MgSO4, 10 mM HEPES, and 5.5 mM D- glucose. All solutions were adjusted to pH 7.4 and continuously bubbled with 95%

O2/5% CO2. At the beginning of experiments, tubules were bathed and perfused with the Cl--containing solution.

To identify principal and intercalated cells, we labeled the apical membrane of intercalated cells by adding fluorescent peanut lectin (PNA, Vector Labs) to the luminal perfusate for 5 minutes and observed which cells were fluorescent. Intracellular pH in CCD cells was assessed with imaging-based, dual excitation- wavelength fluorescence microscopy with use of the fluorescent probe 2',7'-Bis- (2-Carboxyethyl)-5- (And-6)- carboxyfluorescein (BCECF, Molecular probes). Tubules were loaded for ~20 min at room temperature with 5x10-6mol/l of the acetoxymethyl ester of BCECF added to the peritubular fluid. Loading was continued until the fluorescence intensity at 440 nm excitation wavelength was at least one order of magnitude higher than background fluorescence. The loading solution was then washed out by initiation of bath flow and the tubule was equilibrated with dye-free solution for 5-10 minutes. Bath solution was delivered at a rate of 20 ml/min and warmed to 37 ± 0.5°C by water jacket immediately

37 upstream to the chamber. During the fluorescence recording, the Cl-containing solution was delivered to the perfusion pipette via a chamber under an inert gas

(N2 pressure around 1 bar) connected through a manual 6-way valve. With this system, opening of the valve instantaneously activated flow of one solution. The majority of the fluid delivery to the pipette exited the rear of the pipette system through a drain port at a rate of 4 ml/min. This method resulted in a smooth and complete exchange of the luminal solution in less than 3 to 4 sec. as measured by the time necessary for appearance of colored dye at the tip of the perfusion pipette. After 3-minutes recording, luminal fluid was instantaneously replaced by the corresponding Cl-free solution for 3 minutes. Finally, the luminal solution was changed again for the Cl-containing solution.

Intracellular dye was excited alternatively every 2 seconds at 440 and 500 nm with a light-emitting diode (Optoled, Cairn Research, Faversham, UK). Emitted light was collected through a dichroïc mirror, passed through a 530 nm filter and focused onto an EM-CCD camera (iXon, Andor Technology, Belfast, Ireland) connected to a computer. The measured light intensities were digitized with 14-bit precision (16384 grey level scale) for further analysis. For each tubule, 3-4 intercalated cells were analyzed and the mean grey level was measured with the Andor IQ software (Andor Technology, Belfast, Ireland). Background fluorescence was subtracted from fluorescence intensity to obtain intensity of intracellular fluorescence.

Intracellular dye was calibrated at the end of each experiment using the high [K+]- nigericin technique. Tubules were perfused and bathed with a HEPES-buffered, 95-mM K+ solution containing 10 µM of the K+/H+ exchanger nigericine. Four different calibration solutions, titrated to pH 6.5, 6.8, 7.2 or 7.5 were used.

Initial rates of pHi change (dpHi/dt) were determined by a computer-assisted fitting of the pHi versus time data to a linear regression line over the first 15-30 seconds.

3.5 Measure of the fractional volume

The fractional volume if CCDs was measure by an in silico analysis, as described by Mutig et al, 2011 (Mutig, Kahl et al. 2011).

3.6 Measurements of blood pressure by radiotelemetry

The catheter of the telemeter was inserted into the left carotid artery. The transmitter probe was positioned subcutaneously on the flank. After a one-week recovery period in individual cages, mice were placed on a receiver and blood pressure and locomotor activity were recorded continuously in freely moving mice, in a light/dark-cycled recording room (7am to 7pm), for 3 consecutive days on a normal diet as previously described (Trepiccione, Soukaseum et al. 2017).

3.7 Immunoblotting

Animals were sacrificed by cervical dislocation. Kidneys were removed and placed in ice- cold NaCl 0.9%. Cortex was excised under a stereomicroscope and placed into ice-cold isolation buffer (250 mM sucrose, 20 mM Tris-Hepes, pH 7.4) enriched with protease inhibitor cocktail (Roche), anti-phosphatase inhibitor cocktail (Roche), and pepstatin A (1.5 mg/ml). Cortical minced tissues were homogenized in 1 ml homogenization buffer, by using Fast Prep 24 5G sample preparation system (MP Biomedicals) at 5 m/sec speed for 15 sec, twice.

The homogenate was centrifuged at 4000 g for 15 min at 4°C, and the supernatant was centrifuged at 17,000 g for 30 min at 4°C. The pellet was resuspended in isolation buffer. Protein concentration were determined using the Bradford protein assay (Bio Rad Laboratories, Hercules, CA).

Membrane proteins were solubilized in SDS-loading buffer (62.5 mM Tris HCl, pH 6.8, 2% SDS, 100 mM dithiotreitol, 10% glycerol and bromophenol blue), and incubated at room temperature for 30 min. Electrophoresis was initially performed for all samples on 7.5% polyacrylamide minigels (XCell SureLock Mini-Cell; Invitrogen, Carlsbad, CA), which were stained with Coomassie blue to provide quantitative assessment of loading, as previously described (Gueutin V, J Clin Invest 123: 4219– 4231, 2013).

For immunoblotting, proteins were transferred electrophoretically (XCell II Blot Module, Invitrogen Life Technologies) for 2 h at 4°C from unstained gels to nitrocellulose membranes (Amersham) and then stained with 0.5% Ponceau S in acetic acid to check uniformity of protein transfer into the nitrocellulose membrane.

Membranes were first incubated in 5% non-fat dry milk diluted in PBSpH 7.4 for 1 h at room temperature to block nonspecific binding of antibody, followed by overnight at 4°C with the primary antibody (anti-NCC; anti-NCC phospho-Thr53, anti-Pendrin, anti-α and -γ ENaC, were used in a dilution of 1:10000; anti-NDCBE was used 1:250 and anti-ROMK in 1:2000, in PBS containing 1% non-fat dry milk. After five 5 min washes in PBS containing 0.1% Tween-20, membranes were incubated with 1:10000 dilution of goat anti-rabbit IgG (Bio-Rad) conjugated to horseradish peroxidase in PBS containing 5% non-fat dry milk for 2 hours at room temperature. Blots were washed as above, and luminol-enhanced chemiluminescence (ECL, Perkin Elmer Life Science Products) was used to visualize bound antibodies. Detection of chemiluminescence was performed using the mini-LAS imaging system (Fuji). Quantification of each band was performed by densitometry using Multi Gauge software. Densitometric values were normalized to the mean for the control group that was defined as 100% and results were expressed as mean ± SEM.

3.8 Real-time quantitative RT-PCR

Total RNA from whole kidney tissue was extracted in Trizol reagent (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's instructions. Total RNA was treated with deoxyribonuclease I treatment (Ambion, Life Technologies, Carlsbad, CA), according to the specific protocol and followed by inactivation with DNAse Inactivator (Ambion, Life Technologies, CA). The RNA conectration was measured by spectrophotometer and verified by Qubit fluorometer (Invitrogen, Life Technologies, Carlsbad, CA, USA). 500 ng of total RNA were reverse transcribed using iScript Reverse Transcription Supermix (Biorad, Hercules, CA) and random primers provided in the iSript kit for a complete RNA coverage transciptase. The quantitative Real-time RT-PCR (RT-qPCR) was carried out using SYBRgreen iTaq Universal SYBR® Green Supermix (Biorad, Hercules, CA) on a CFX96 Real Time System thermal cycler (Biorad, Hercules, CA), according to the manufacturer´s instructions. Controls without template were included to verify the specificity on the fluorescence to avoid dimer formation or PCR contaminations. The specificity of each primer was analyzed using the melting curve to confirm that a single amplicon was generated. Standard curves for each gene were generated using

40 serial dilutions from a cDNA pool of all samples. Normalization for reverse transcriptase efficiency and RNA quantity was performed in accordance with the MIQE guidelines (Bustin, Benes et al. 2009) against four different reference genes of different expression levels (geometric mean of the expression of Ribosomal 18S RNA, HPRT, GAPDH and Ubiquitin). The relative expression ratio of renin in mutants vs. control animals was calculated taking into account the PCR efficiency (Pfaffl 2001). Primer sequences for renin were: Renin forward 5´- ATGAAGGGGGTGTCTGTGGGG-3´ and reverse 5´- ATGTCGGGGAGGGTGGGCACCTG-3´; Ubiquitin forward 5´- AGCCCAGTGTTACCACCAAG-3´, reverse 5´- ACCCAAGAACAAGCACAAGG- 3´ ; GAPDH forward 5´-AAGGTCATCCCAGAGCTGAA-3´, reverse 5´- CTGCTTCACCACCTTCTTGA-3´ ; HPRT forward, 5´- CACAGGACTAGAACACCTGC-3´, reverse 5´-GCTGGTGAAAAGGACCTCT-3.

3.9 Intercalated cell type quantification

For immunostaining, mice were anesthetized by intraperitoneal injections of ketamine (80 mg/kg)/ xylazine (10 mg/kg) and perfused by aorta artery with 4% of PFA. The kidneys were submitted to fast freezing in isopentane. Cryosections (4 um) were blocked with 10% NGS + 0.05% of BSA in PBS for 1h at room temperature and the corresponding primary antibody was incubated overnight in PBS 0.05% BSA at 4°C. Afterward the secondary antibody were incubate during 1 hour at room temperature in 0.05% of BSA in PBS.

Kidney sections from controls and TgWnk4PHAII mice were co-stained with either AE1, E subunit of the V-ATPase or pendrin respectively. Randomly photographed cortex areas (n=12 fields) were analyzed for co-expressing cells by blinded experimenters and the total number of ICs was calculated with standard error as the sum of both sub-populations.

3.10 Antibody sources

Antibodies against alpha and gamma subunits of ENaC, and phospho-T53 NCC were a kind gift from Dr. Johannes Loffing (University of Zürich, Zürich, Switzerland). Anti-NCC antibody was a gift from Dr. David Ellison (Oregon Health

41 and Science University, Portland, OR). Antibody against pendrin was a gift from Dr. Peter Aronson (Yale University, New Haven, CN). Antibody against the E subunit of the V-ATPase was kindly provided by Dr. Denis Brown (Harvard Medical School, Boston, MA). Anti AE1 antibody was provided by Carsten Wagner (University of Zurich, Switzerand). ROMK antibody was provided from Paul Welling (University of Maryland, Baltimore). Antibody against NDCBE is from Synaptic Systems.

3.11 In vitro microperfusion of mouse CCDs and transepithelial ion flux measurement

As previously reported (Gueutin, Vallet et al. 2013), kidneys were removed and cut into 1- to 2-mm coronal slices that were transferred into a chilled dissection medium containing (in mM): 118 NaCl, 25 NaHCO3, 2.0 K2HPO4, 1.2 MgSO4, 2.0 calcium lactate, 1.0 sodium citrate, 5.5 glucose, and 12 creatinine, pH 7.4, and gassed with 95% O2/5% CO2. CCD segments were isolated from cortico-medullary rays under a dissecting microscope with a sharpened forceps. Because CCDs are highly heterogeneous, relatively short segments (0.45–0.6 mm) were dissected to maximize the reproducibility of the isolation procedure. In vitro microperfusion was performed as described by Burg et al. (Burg, Grantham et al. 1966).Isolated CCDs were rapidly transferred to a 1.2-ml temperature and environment controlled chamber, mounted on an inverted microscope, and then perfused and bathed initially at room temperature with dissection solution. The specimen chamber was continuously suffused with 95%O2 / 5%CO2 to maintain pH at 7.4. Once secure, the inner perfusion pipette was advanced, and the tubule was opened with a slight positive pressure. The opposite end of the tubule was then pulled into a holding collection pipette. In the holding collection pipette, 2–3 cm of water-saturated mineral oil contributed to maintain the tubule open at a low flow rate of perfusion. The perfusing and collecting end of the segment was sealed into a guard pipette using Dow Corning 200 dielectric fluid (Dow Corning Corp.). The tubules were then warmed to 37°C and equilibrated for 20 minutes while the collection rate was adjusted to a rate of 2 nl/min. The length of each segment was measured using an eyepiece micrometer. Because CCDs from mice are frequently unstable and collapse rapidly, measurements were conducted during the first 90 minutes of

42 perfusion. Usually, collections from 4 periods of 15 minutes were performed in which 25 to 30 nanoliters of fluid were collected. The volume of the collections was determined under water-saturated mineral oil with calibrated volumetric pipettes. For [Na+], [K+] and [creatinine] measurements, 20 nl were required, while 2–3 nanoliters were used for [Cl-] determinations.

[Na+] and [creatinine] measurements were performed by HPLC and [Cl-] were measured by microcoulometry as previously described (Leviel, Hubner et al. 2010).

Creatinine was used as the volume marker and therefore was added to the perfusion solutions (both perfusate and bath) at a concentration of 12 mM. The rate of fluid absorption (Jv) was calculated as Jv = (Vperf – Vcoll) / L, with Vperf =

Crcoll / Crperf x Vcoll, where Crcoll and Crperf are the concentrations of creatinine in the collected fluid and perfusate, respectively, Vcoll is the collection rate at the end of the tubule, and L is the length of the tubule.

+ For each collection, Na flux (JNa) was calculated and reported to the length of the tubule: JNa = [([Na]perf x Vperf) – ([Na]coll x Vcoll)] / L and JCl = [([Cl]perf x Vperf) – ([Cl]coll x Vcoll)] / L, JK = [([K]perf x Vperf) – ([K]coll x Vcoll)] / L, where perf indicates perfusate and coll indicates collection fluid. Therefore, positive values indicate net absorption, whereas negative values indicate net secretion of the ion. For each tubule, the mean of the 4 collection periods was used.

3.12 Statistics and data analysis

Data are expressed as means ± SEM for each experiment. One-way t-test or one- way ANOVA were performed to analyze the difference between groups. The data were significant for P ≤ 0.05. Scatterplots and all comparisons of means were

analyzed with Prism software (version 6).

3.13 Study approval

All the experimental procedures were conformed to the “Protocol of Animal Welfare” (Amsterdam Treaty; www.eurocbc.org/page673.html) and were approved by the French Government animal welfare policy (Agreement number A75-15-32).

4. RESULTS

4.1 TgWnk4PHAII mice exhibit hyperkalemic metabolic acidosis

Dr. Richard Lifton from Yale University provided the TgWnk4PHAII mouse used in this study (Lalioti, Zhang et al. 2006). This mouse model is carrying a WNK4 missense mutation (Q562E) identified in PHAII patients.

TgWnk4PHAII mice as was expected and already shown by others (Lalioti, Zhang et al. 2006, Yang, Morimoto et al. 2007), display a remarkable increase in the protein abundance of NCC and its phosphorylated form (phosphor-T53 NCC) (Figure 8).

Figure 8. Protein abundance of NCC and pNCC is increased in TgWnk4PHAII mice. Inmmunoblots for NCC and its T53 phosphorylated residue (pNCC) on plasma membrane-enriched fractions from renal cortex of TgWnk4PHAII (n=6) and control (n=4) mice. Bar graph shows a summary of densitometric analyses. Densitometric values were normalized to the mean for the control group that was defined as 100% and results were expressed as mean ± SEM. Statistical significance was assessed by unpaired Student’s t-test. **P<0.001

Furthermore, TgWnk4PHAII mice exhibit all the phenotypic abnormalities characterizing PHAII: metabolic hyperchloremic acidosis and hyperkalemia, as was revealed by the analysis of blood parameters (Figure 9A-E). In addition, mRNA abundance of renin is significantly lower in the PHAII mice (Figure 9F), while urinary aldosterone excretion was significantly increased in TgWnk4PHAII mice (Figure 9G), in accordance with the observation in most of PHAII patients.

A B C

D E F G

Figure 9. TgWnk4PHAII mice exhibit phenotypic abnormalities characteristic of PHAII. A-E. The indicated blood parameters were measured in control (white bars) and TgWnk4PHAII mice (back bars), n=8 mice per group. F. Renin gene expression was analyzed by quantitative RT-PCR in whole kidney of control (white bars) and TgWnk4PHAII mice (black bars). Results are expressed in arbitrary units relative to the expression of a geometrical mean of 4 housekeeping genes: ubiquitin, hrp, 18s and GAPDH. The control group has been arbitrarily set to 1, n= 6 mice per group. G. Urinary aldosterone excretion was measured in control (white bars) and TgWnk4PHAII mice (black bars), the values were normalized by the creatinine concentration measured in the same samples (in mM), n=8 for control and n=7 for TgWnk4PHAII mice. All the values are represented as means ± SEM and statistical significance was assessed by unpaired Student’s t-test. *P<0.05, **P<0.01, and ***P<0.001.

4.2 Electroneutral thiazide-sensitive NaCl transport is activated in the collecting duct of TgWnk4PHAII mice

It has been established that under PHAII condition the activity on NCC in the DCT is dramatically upregulated. However, the specific contribution of the CCD where the final regulation of sodium excretion takes place, to the PHAII phenotype has not been explored in deep so far.

As previously described by Leviel, et al in 2010, under basal condition, no transport activity is detectable in CCDs isolated from wild-type mice when they are microperfused in vitro. However, once the mice were fed with sodium depleted diet during 2 weeks before the microperfusion experiment, it was possible to observe absorption of Na+ and Cl-, associated to secretion of K+, with the generation of a lumen-negative transepithelial voltage, consistent with ENaC-mediated Na+ absorption. In addition to the electrogenic pathway, the authors described electroneutral thiazide-sensitive sodium reabsorption, mediated by the Na+-driven - - + - - Cl /HCO3 exchanger (NDCBE) and the Na -independent Cl /HCO3 exchanger (pendrin).

In order to analyze the effects of the PHAII-causing mutation of WNK4 Q562E on + + - CCD ion transport, transepithelial fluxes of Na (JNa), K (JK), Cl (JCl) and the transepithelial voltage (Vte) were measured in microperfused CCDs isolated from TgWnk4PHAII mice fed a normal sodium diet (0,3% Na+).

CCDs isolated from TgWnk4PHAII mice exhibited net NaCl absorption but did not

+ develop a significant lumen negative (Vte) and did not significantly secrete K , suggesting the absence of detectable ENaC activity (Figure 10, black bars). Interestingly, NaCl absorption was fully inhibited by luminal addition of 100 µM hydrochlorothiazide (HCTZ) (Figure 10, striped bars). These experiments demonstrate that beside the increased NCC activity, the electroneutral NaCl transport mediated by pendrin/NDCBE is stimulated by WNK4 PHAII-causing mutation, contributing to the excess of NaCl reabsorption.

Figure 10. Electroneutral NaCl transport system is activated in the CCD of PHAII + - + TgWnk4 mice. Transepithelial fluxes of Na (JNa), Cl (JCl), K (JK) and transepithelial voltage (Vte) were measured in microperfused CCDs isolated from TgWnk4PHAII mice, fed on normal diet in the absence (Black bars, n=5) or presence of luminal hydrochlorothiazide (HCTZ, striped bars, n=6). Statistical analysis was performed by student t test. *P<0.05.

4.3 ENaC-dependent K+ secretion is blocked in TgWnk4PHAII mice despite unchanged renal ENaC activity

In the complex phenotype of PHAII patients, the associated hyperkalemia remain unexplained despite many hypotheses. The increased NaCl absorption in the DCT reduces the downstream Na+ delivery to the CNT and CD. In turn, this process could lower ENaC activity and affect lumen negative transepithelial voltage, which is necessary for the proper excretion of K+ through ROMK. Accordingly, we did not detect any ENaC activity in the microperfusion experiments presented in Figure 10.

Importantly, ENaC is not only present in CCD but also in CNT, a nephron segment, which is not suitable for in vitro microperfusion. Thus, to have an overall idea of ENaC protein abundance and activity under PHAII phenotype, western blot experiment of the α and γ subunits of ENaC were performed, using plasma membrane-enriched preparations isolated from renal cortex of TgWnk4PHAII and control mice.

The abundance of the full length 90 kDa form of α-ENaC and its cleaved 30 kDa fragment were increased in TgWnk4PHAII mice. Also, the abundance of the cleaved 70 kDa band of γ-ENaC was increased while the full length version of γ-ENaC was decreased in TgWnk4PHAII mice (Figure 11). The abundance profile of the different ENaC subunits is associated with the level of aldosterone (Masilamani, Kim et al. 1999). Thus, the increase in α-ENaC expression and that of the proteolytic cleavage of γ-ENaC are both consistent with increased aldosterone secretion in TgWnk4PHAII mice as shown in Figure 9G.

Figure 11. α-ENaC and the proteolytic cleavage form of γ-ENaC are increased in TgWnk4PHAII mice. α-ENaC and γ-ENaC protein abundance was assessed by western blot of plasma membrane-enriched preparations obtained from the renal cortex of control (n=4) and TgWnk4PHAII (n=6) mice. Bar graph shows a summary of densitometric analyses. Densitometric values were normalized to the mean for the control group that was defined as 100% and results were expressed as mean ± SEM. Statistical significance was assessed by unpaired t test. *P<0.05, **P<0.01

Additionally, in order to estimate the global ENaC activity in PHAII mice, an acute amiloride injection was used to block ENaC and to evaluate its effect on urinary Na+ and K+ excretion in TgWnk4PHAII and control mice. Despite the proteolytic ENaC activation, the natriuretic response to amiloride was identical in control and mutant mice (Figure 12A), suggesting that Na+ reabsorption through ENaC is not altered in TgWnk4PHAII mice. However, amiloride had significantly less effect on urinary K+ excretion in TgWnk4PHAII mice than in control mice (Figure 12B), demonstrating that ENaC-dependent K+ transport mechanisms per se are inhibited in TgWnk4PHAII.

A B

Figure 12. Identical natriuretic response to amiloride observed in TgWnk4PHAII and control mice. Effect of amiloride injection on urinary Na+ (A) and K+ excretion (B) in control and TgWnk4PHAII mice. Mice kept under standard diet were subcutaneously injected with either vehicle or amiloride (1.45 mg/kg body weight). Urines from control and TgWnk4PHAII mice were collected over 2 consecutive days starting at 10 AM and ending at 4 PM. Vehicle was injected on day 1 (black bars), whereas amiloride was injected at 10 AM on the second day (white bars). Excreted Na+ and K+ concentrations (mM) were normalized to the urinary concentration of creatinine (mM) to minimize the effects of incomplete urine sampling over such short periods. Values are the mean ± SEM from 8 determinations in each group of mice. Statistical significance was assessed by one-way ANOVA. ***P<0.001 and ****P<0.0001, vehicle vs. amiloride. *P<0.05, control vs. TgWnk4PHAII following amiloride injection.

However, despite the difference in K+ excretion observed after amiloride injection between the two types of mice, immunoblots revealed the same level of ROMK protein expression in TgWnk4PHAII mice and control, and a small increase in the 2+ + abundance of α-BKCa (α subunit of the large-conductance Ca -activated K channel), which is expressed along with ROMK. This might indicate a compensatory effect in order to reduce the hyperkalemia present in TgWnk4PHAII mice (Figure 13).

Figure 13. Slight increase in α-BKCa and not changes in ROMK protein abundance in TgWnk4PHAII mice. Immunoblots for α-BKCa and ROMK were performed on plasma membrane-enriched preparations of the renal cortex of TgWnk4PHAII (n=6) and controls (n=4). Densitometric values were normalized to the mean for the control group that was defined as 100% and results were expressed as mean ± SEM. Statistical significance was assessed by unpaired t test. *P<0.05.

4.4 Normalization of plasma K+ concentration does not correct metabolic acidosis in TgWnk4PHAII mice

The above result showed an impaired potassium secretion by the distal nephron despite a normal ENaC function, in line with the hyperkalemia present in TgWnk4PHAII mice and in patients. Hyperkalemia can contribute to the development of renal tubular acidosis (DuBose 1997) by inhibiting the NH3 transport in the proximal tubule and thick ascending limb, and also by blocking NH3 production in the proximal tubule (Karet 2009). In order to determine the importance of high plasma potassium concentration in the development of acidosis in PHAII, we tested whether normalization of plasma potassium concentration can alleviate the acidosis of TgWnk4PHAII mice. TgWnk4PHAII mice were fed with a synthetic low K+ diet during four days. As expected, plasma K+ concentration [K+] was normalized following this treatment (Figure 14). However, the decrease in plasma [K+] did not

- modify either plasma HCO3 concentration or pH, indicating that acidosis in PHAII is not caused by hyperkalemia.

Figure 14. Low potassium diet decreases blood plasma K+ concentration in PHAII - TgWnk4 mice without modifying either blood pH or blood HCO3 concentration. Blood gas parameters were measured in TgWnk4PHAII mice fed a normal diet and after four days on low K+ diet. Values are the mean ± SEM from 7 determinations in each group of mice. Statistical analysis was performed by paired t-student. NS, not significant, P>0.05.

4.5 Renal acidosis in TgWnk4PHAII mice is not caused by impaired proton secretion by the distal nephron

To elucidate the mechanism behind of the metabolic acidosis present in TgWnk4PHAII mice, it is also necessary to investigate a possible impaired proton secretion by the distal nephron, as occur in distal renal acidosis (type I) because of an impaired function of vH+-ATPase (Stehberger 2003). Thus, an acid load was administrated as NH4Cl (0.28 M) in the drinking water during 15 days to TgWnk4PHAII and control mice.

- Figure 15 shows that before acid loading, only plasma HCO3 concentration (Figure 15B) was significantly lower in TgWnk4PHAII mice, while urine pH (Figure 15C), urinary titrable acid excretion (Figure 15D) and ammonium excretion (Figure 15E) were identical in TgWnk4PHAII and control mice. After 2 days of acid loading, - blood pH and HCO3 concentration decreased in both groups. However, TgWnk4PHAII mice developed a much stronger metabolic acidosis than control mice. When acid loading was continued, control mice were able to cope with the

- acid load and exhibited normal blood pH and HCO3 concentration at day 15 of the treatment. By contrast, severe metabolic acidosis persisted in TgWnk4PHAII mice. Interestingly, following acid loading, urine pH decreased and ammonium excretion increased maximally in both groups indicating that proton secretion and ammonium transport (or metabolism) are normal in TgWnk4PHAII mice.

Furthermore, figure 15F shows a marked shift in the relationship between ammonium urinary excretion and blood bicarbonate concentration (reflecting the severity of metabolic acidosis) in TgWnk4PHAII vs. control mice. This demonstrated that, while proton secretion or ammonium excretion capabilities are preserved in TgWnk4PHAII mice, these mice clearly have a defect in renal net acid excretion. This led us to hypothesize that metabolic acidosis in PHAII is not caused by impaired acid excretion but is rather due to increased renal loss of base.

Figure 15. Metabolic acidosis in TgWnk4PHAII mice is not caused by impaired acid excretion. Time course of blood parameters analysis (A-B) and urinary acid- base homeostasis parameters (C-E) from control (white bar and open circle) and TgWnk4PHAII mice (black bar and black circle), during 15 days of acid load (280 mM of NH4Cl in drinking water). Values are the mean ± SEM from 8 + determinations in each group of mice. Relationship between urinary NH4 and - plasma bicarbonate concentration [HCO3 ] before and after the acid load (F). Values of blood and urinary parameters in TgWnk4PHAII mice and in controls are represented as means ± SEM. Statistical significance was assessed by unpaired t test. *P<0.05, **P<0.01).

The effect of acid loading was also tested in WNK1PHAII mice, another PHAII model in which the first intron of WNK1 is deleted, leading to an increase of the WNK1 protein abundance (Delaloy, Elvira-Matelot et al. 2008). When submitted to an acid load (Figure 16), we observed that the mice responded exactly like TgWnk4PHAII mouse. Therefore, suggesting that the same mechanism is probably causing acidosis in both PHAII models independently of the mutation.

Figure 16. Response to an acid load in WNK1PHAII mice indicates a normal - - acid excretion. Blood pH, pCO2, Cl and HCO3 concentration were measured in WNK1∆i1 and in control mice (WNK1lox), in basal conditions and after administrating 280 mM of NH4Cl in the drinking water for 7 days. Urine pH and + urinary NH4 were also measured at basal condition (day 0) and at days 2 and 7, statistical significances are represented respect to day 0. Values are represented as mean ± SEM. Statistical significance is assessed by one-way ANOVA n=8 in both groups, *p<0.05, **p<0.01, ***p<0.001.

4.6 Renal acidosis in TgWnk4PHAII mice is a consequence of an increased of the pendrin activity

As the renal acidosis observed in TgWnk4PHAII mice, is not a consequence of hyperkalemia and is not due to an impaired proton secretion in the distal nephron, - - we investigated whether base excretion through Cl -HCO3 exchanger pendrin in β- intercalated cells is working properly under WNK4-PHAII mutation.

To assess this, pendrin activity was determined by measuring Cl--dependent alkalinization in intercalated cells of CCDs isolated from TgWnk4PHAII and control mice (Figure 17A). The rate of intracellular alkalinization in response to luminal Cl- - - removal, considered as an estimate of the apical Cl /HCO3 exchange activity, was much higher in intercalated cells from TgWnk4PHAII mice than in control mice (Figure 17B). These results indicate that pendrin activity per cell is increased in TgWnk4PHAII mice.

A 7.2 - B ) [Cl ]perf = 0 c

7.0 / 6 *

i 6.8 n

6.6 0

( 2

6.4 PHAII t

TgWnk4 d

/ Control i 6.2 H 0

60 s d

- - Figure 17. Increased apical Cl /HCO3 exchange activity in β-intercalated cells of TgWnk4PHAII mice. A. Representative profile of intracellular pH changes following luminal chloride removal in intercalated cells in isolated CCDs from control (black curve) and TgWnk4PHAII mice (red curve). B. Average initial rates of pHi recovery after chloride removal in intercalated cells from control (white bars, n=4) and TgWNK4PHAII (black bars, n=7). Values are represented as means ± SEM, *P<0.05, unpaired t test.

- - In order to correlate the higher apical Cl /HCO3 exchange activity observed in β- ICs with the protein abundance of NDCBE and pendrin, semi-quantitative immunoblotting of plasma membrane enriched preparations from kidney cortex of TgWnk4PHAII and control mice were performed. The protein abundance of NDCBE and pendrin were unchanged (Figure 18A). Nevertheless, pendrin abundance is regulated primarily through changes in subcellular distribution, rather than by changes in total protein abundance (Verlander, Hassell et al. 2003). Further, immunofluorescence experiments revealed an increase of pendrin labeling in the collecting ducts located in renal medullary rays of TgWnk4PHAII mice (Figure 18B).

As described previously, ICs are classified in 3 different forms according to the transporters that they express: (1) α-ICs characterized by apical expression of the V-ATPase and basolateral expression of an anion exchanger AE1, (2) β-ICs which harbor apical pendrin and basolateral V-ATPase, and (3) non α-, non β-ICs with apical pendrin and apical V-ATPase. Although the total number of ICs remains generally constant, the relative number of α-ICs vs. β-ICs is tightly controlled and adapted depending on the acid-base status. For instance, metabolic acidosis by promoting transdifferentiation of β-ICs into α-ICs significantly increases the number of α-ICs while a converse decrease in β-ICs is observed (Schwartz, Barasch et al. 1985). Thus, to determine whether this phenomenon is altered in PHAII mice, the number of the different IC types in the CCDs of control and TgWnk4PHAII mice was evaluated. In sections labeled for pendrin, E subunit of the V-ATPase and AE1, intercalated cells were defined as cells stained for the E subunit of the V-ATPase; α-ICs were defined as those that also exhibited basolateral AE1 staining, whereas β-IC and non α-, non β-ICs were identified by the presence of pendrin staining. In both transgenic and control mice, CCDs located in medullary rays exhibited α- and β-ICs but no non α-, non β-ICs.

In the control mice, 1867 intercalated cells were counted from 4 independent animals (Figure 18C, white bars). 37% of ICs were positive for pendrin, whereas 63% of cells were positive for AE1. In 4 independent TgWnk4PHAII mice, 1955 were intercalated cells (Figure 18C, black bars), with a fraction of β-ICs significantly increased since we observed 50% of ICs were pendrin-positive. Conversely, the number of α-ICs was reduced and the total number of ICs was not different between control and TgWnk4PHAII mice. Considering that pendrin activity per cell is

57 increased in TgWnk4PHAII mice (Figure 17), we can conclude that total pendrin activity is increased in CCDs of mutant mice, although there was no change observed in the total protein abundance (figure 17A). As shown in Figure 18D, this increased pendrin transport activity was associated with a significant increase in the fractional volume of the CCD from TgWnk4PHAII mice (1.62 ± 0.21 % of kidney cortex in control mice vs. 2.33 ± 0.13 % of kidney cortex in mutant mice, n=4 for each group, p=0.031).

Figure 18. The number of β-intercalated cells in TgWnk4PHAII mice is increased. A. Immunoblots for pendrin and NDCBE were performed on plasma membrane-enriched preparations of the renal cortex of TgWnk4PHAII (n=6) and controls (n=4). Densitometric values were normalized to the mean for the control group that was defined as 100% and results were expressed as mean ± SEM. B. Immunofluorescence of collecting ducts in medullary rays from controls and TgWnk4PHAII mice. E subunit of the V-ATPase (blue) was used as a marker of CCDs. β-ICs were identified as apical pendrin positive cells (red) and α-ICs were defined by the basolateral AE1 staining. C. Fraction of α-IC cells and β-ICs cells in relation of total intercalated cells in CCD. Control mice (white bars) and TgWnk4PHAII (black bars), n=4 for both group. *P<0.05, unpaired t test. D. Fractional volume of the CCD respect to the kidney cortex from TgWnk4PHAII mice (black bars) and control mice (white bars), n=4 for each group, results were expressed as mean ± SEM. p=0.03, unpaired t test.

4.7 Hyperchloremic metabolic acidosis and hyperkalemia in TgWnk4PHAII mice are corrected by pendrin genetic ablation.

According to above experiments, we can therefore conclude that pendrin is hyperactive in TgWnk4PHAII mice. To assess whether pendrin is contributing to PHAII phenotype, TgWnk4PHAII mice were bred with a pendrin KO mouse (Everett, Belyantseva et al. 2001).

Double transgenic mice (TgWnk4PHAII/Pds-/-) no longer exhibited hyperchloremic metabolic acidosis and hyperkalemia, as demonstrated by the increase of bicarbonate, reduction of chloride and potassium concentration in blood (Table 2).

Table 2: Blood parameters of TgWNK4PHAII and TgWNK4PHAII /Pds-/- mice.

Blood TgWNK4PHAII TgWNK4PHAII ;Pds-/- pH 7.30 ± 0.01 7.34 ± 0.02

PCO2 51 ± 2 53 ± 3 - [HCO3 ], mM 22.5 ± 0.3 25.7 ± 0.8** [Na+], mM 152.6 ± 0.7 151.3 ± 0.6 [Cl-], mM 116.3 ± 0.6 113.7 ± 0.7* [K+], mM 4.46 ± 0.09 3.91 ± 0.07*** Hematocrit, % 38.9 ± 1.1 41.2 ± 0.6* Values are means ± SEM, n=9 for TgWNK4PHAII and n=10 for TgWNK4PHAII/Pds-/-. *P<0.05, **P<0.01, and ***P<0.001.

These results confirmed that activation of pendrin is involved in the onset of hyperchloremic metabolic acidosis and hyperkalemia characteristic of PHAII phenotype. The effect of pendrin genetic ablation is reminiscent of that of thiazides, drug able to block the electroneutral Na+ reabsorption processes in both the DCT and the CCD. Indeed, a decrease of blood K+ concentration and an increase of blood bicarbonate concentration were observed in TgWnk4PHAII mice (Table 3), rapidly after an injection of thiazide (6 hours).

Table 3: Blood parameters of TgWNK4PHAII before and 6h after thiazide injection.

Blood Before HCTZ After HCTZ pH 7.28 ± 0.01 7.36 ± 0.02*

PCO2 47 ± 2 49 ± 6 - [HCO3 ], mM 20.4 ± 0.7 25.0 ± 0.8*** [Na+], mM 149.9 ± 0.7 148.0 ± 0.6 [Cl-], mM 114.1 ± 0.9 106.0 ± 0.6**** [K+], mM 4.55 ± 0.12 3.48 ± 0.07**** Hematocrit, % 37.0 ± 1.3 45.1 ± 0.8*** Values are means ± SEM *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001; n=7 for both groups.

Additionally, pendrin disruption also led to a significant increase in renin mRNA levels in TgWnk4PHAII mice (1.00 ± 0.1, n=7 in TgWnk4PHAII vs. 1.92 ± 0.45, n=6 in TgWnk4PHAII/Pds-/- mice, P<0.01), in association to a higher hematocrit (table 2). However, no difference between groups was detected in blood pressure measured by telemetry (123.7 ± 1.2 mmHg in TgWnk4PHAII/Pds-/- mice, n=5 vs. 128.8 ± 2.4 mmHg in TgWnk4PHAII mice, n=6, NS) or in aldosterone levels (7.26 ± 0.57 nM/mM creatinine in TgWnk4PHAII/Pds-/- mice, n=10 vs. 7.93 ± 0.63 nM/mM creatinine in TgWnk4PHAII mice, n=12, NS).

Thus, the effects of pendrin disruption on vascular volume and blood pressure seem to remain limited, presumably because NCC is still markedly stimulated in TgWnk4PHAII/Pds-/- mice. Accordingly, the expression of the various Na+ transporters of the distal nephron was assessed by semi-quantitative immunoblotting of plasma membrane enriched preparations from kidneys of TgWnk4PHAII and TgWnk4PHAII/Pds-/- mice (Figure 19). No change in protein abundance of NCC and phosphor-T53 NCC were observed between double transgenic and TgWnk4PHAII mice. Protein abundance of α and γ subunits of ENaC were decreased in TgWnk4PHAII/Pds-/- mice when compared to TgWnk4PHAII mice, consistent with the effect of pendrin deletion on ENaC expression previously described by others (Kim, Pech et al. 2007).

Figure 19. Protein abundances of different Na+ transporters in TgWnk4PHAII and TgWnk4PHAII/Pds-/- mice. Immunoblots performed on membrane-enriched fraction of kidney from TgWnk4PHAII (n=6) and TgWnk4PHAII/Pds-/- (n=6). Bar graph shows a summary of densitometric analyses. Densitometric values were normalized to the mean for the TgWnk4PHAII mice that was defined as 100% and results were expressed as mean ± SEM. Statistical significance was assessed by unpaired Student’s t-test. *P<0.05, **P<0.01, ***P<0.001.

According to our results, ENaC-dependent K+ secretion was decreased in TgWnk4PHAII mice without any change in ENaC activity suggesting that ROMK itself is not functioning normally, despite we detected no change in its protein abundance (Figure 12 and 13, respectively). Therefore, the total ENaC-dependent Na+ absorption and K+ secretion was estimated in double mutant mice, by assessing the effects of an acute injection of amiloride on urinary excretion of Na+ and K+. The natriuretic response to amiloride (Figure 20A) was not different

62 between TgWnk4PHAII/Pds-/- and TgWnk4PHAII mice indicating that ENaC activity is not altered by pendrin disruption. However, the decrease in urinary K+ excretion following amiloride was higher in TgWnk4PHAII/Pds-/- mice than in TgWnk4PHAII mice (Figure 20B), suggesting that ENaC-dependent K+ secretion is higher after pendrin disruption in TgWnk4PHAII mice.

A B

Figure 20. Effect of amiloride injection on urinary Na+ and K+ excretion in TgWnk4PHAII/Pds-/- and TgWnk4PHAII mice. Mice kept under standard diet were subcutaneously injected with either vehicle or amiloride (1.45 mg/kg body weight). Urines from TgWnk4PHAII and TgWnk4PHAII/Pds-/- mice were collected over 2 consecutive days starting at 10 AM and ending at 4 PM. Vehicle was injected on day 1 (black bars), whereas amiloride was injected at 10 AM on the second day (white bars). Excreted Na+ and K+ concentrations (mM) were normalized to the urinary concentration of creatinine (mM) to minimize the effects of incomplete urine sampling over such short periods. Values are the mean ± SEM from 8 determinations in each group of mice. Statistical significance was assessed by one-way ANOVA. ** P<0.01 and **** P<0.0001.

Consequently, the abundance of ROMK and -BKca were then estimated by western blot. The expression of ROMK is not significantly different in the double transgenic mice in comparison with TgWNK4PHAII. However, -BKca is highly abundant in double transgenic compared with TgWNK4PHAII in agreement to the reduction in blood K+ concentrations (Figure 21).

PHAII Figure 21. α-BKCa and ROMK abundance in TgWnk4 and double PHAII -/- transgenic mice (TgWnk4 /Pds ). Immunoblots for α-BKCa and ROMK were performed on plasma membrane-enriched preparations of kidney of TgWnk4PHAII (n=7) and TgWnk4PHAII/Pds-/- (n=6). Densitometric values were normalized to the mean of TgWnk4PHAII group that was defined as 100% and results were expressed as mean ± SEM. Statistical significance was assessed by unpaired t test. *P<0.05.

5. DISCUSSION

Precise knowledge of the primary cause of a disease is essential for understanding its mechanisms and for adequate classification, prognosis, and treatment. In the case of hypertension, one of the most common diseases and a global health concern, the molecular mechanisms underlying pathogenesis are still poorly understood in the majority of the cases.

Hypertension is a multifactorial pathology, which include from environmental (mainly alimentary habits) to genetic factors. In the present study, we have focused on one hereditary disease named Pseudohypoaldosteronism type II, mainly characterized by hypertension, hyperchloremic metabolic acidosis and hyperkalemia. Hereafter, we present what we know about PHAII and what remains indeterminate, and we discuss how our research contributes to resolve some open questions.

5.1 The NCC upregulation is not sufficient to explain entire the phenotypic features of PHAII

The discovery that mutations in genes encoding two members of the WNK family, WNK1 and WNK4, account for the pathogenesis of PHAII in some patients (Wilson, Disse-Nicodeme et al. 2001); in addition to the mutations of the ubiquitin- ligase complex formed by the proteins KLHL3 and CUL3 (Boyden, Choi et al. 2012, Louis-Dit-Picard, Barc et al. 2012), were essential to the better understanding of physiological changes produced by PHAII.

The fact that all these mutations lead to a gain of function of the thiazide-sensitive NaCl cotransporter NCC of the distal convoluted tubule and the observation that most symptoms of PHAII can be corrected by low dose of thiazide diuretics support the possibility that NCC plays a central role in PHAII, and gives sense to the hypothesis already proposed in 1981 by Schambellan et al., which suggested that the disease is caused by excessive electroneutral absorption of chloride by the distal nephron (Schambelan, Sebastian et al. 1981).

Accordingly, most of the studies conducted so far have been focused on the deregulation of NCC by PHAII-causing mutations. The transgenic mouse used in

65 this study is expressing WNK4 carrying one of the missense mutations identified in PHAII patients (WNK4-Q562E; TgWNK4PHAII mice). The mice display all clinical features of the syndrome ((Lalioti, Zhang et al. 2006) and Figure 8-9). In this model, increase of the NCC phosphorylation and abundance at the apical cell membrane result in enhanced NaCl absorption in the DCT and vascular volume expansion, favoring the onset of hypertension. In addition, WNK4 knock-out mice develop a mild Gitelman-like syndrome, with normal blood pressure, increased plasma renin activity, and reduced NCC expression and phosphorylation (Castaneda-Bueno, Cervantes-Perez et al. 2012). All together these data confirm the importance of NCC in the pathogenesis of PHAII.

However, there are also contradictory results. Surprisingly, a transgenic mouse overexpressing NCC displayed a phenotype similar to wild-type mouse (McCormick, Nelson et al. 2011). Moreover, mice inactivated for KS-WNK1 or Nedd4-2 did not exhibit hyperkalemic hypertension despite increased NCC expression and phosphorylation (Hadchouel, Soukaseum et al. 2010, Ronzaud, Loffing-Cueni et al. 2013).

Finally, in a more recent study, the authors introduced a kinase-activating mutation in the gene codifying for SPAK (Stk39) to drive this constitutively active (CA)- SPAK mutant, specifically to the early DCT (Grimm, Coleman et al. 2017). The mouse model displayed thiazide-treatable hypertension and hyperkalemia, associated to NCC hyperphosphorylation. However, in this model, thiazide- mediated inhibition of NCC restored sodium excretion but not urinary potassium excretion, indicating that NCC is not the sole responsible for the observed PHAII phenotype. Furthermore, CA-SPAK mice exhibited ASDN remodeling, with a reduction in connecting tubule mass, and attenuation of ENaC and ROMK expression. Interestingly, in TgWNK4PHAII model, ENaC attenuation was not detected, but a clear impaired K+ excretion was observed, which was absent in TgWNK4PHAII after pendrin genetic ablation. Thus, an important missing experiment for us, would be to evaluate ASDN remodeling in TgWNK4PHAII in comparison with the double mutant mice.

Overall, this uncertainty leads to the idea that beside NCC other renal transporters are deregulated in PHAII phenotype. According to our results, pendrin is the best candidate.

5.2 Pendrin overactivity is contributing to the pathogenesis of PHAII

Our hypothesis that overactive pendrin might contribute to the pathogenesis of PHAII, is based on previous indirect evidence, showing that pendrin activity may influence long term regulation of blood pressure. For instance, a mouse model overexpressing pendrin in intercalated cells favor the start of chloride-dependent hypertension (Jacques, Picard et al. 2013). Furthermore, pendrin-deficient mice are protected against mineralocorticoid-induced hypertension (Verlander, Hassell et al. 2003) and develop lower blood pressure during NaCl restriction (Wall, Kim et al. 2004). Additionally, a recent study proved that an acute inactivation of pendrin also results in lower blood pressure (Trepiccione, Soukaseum et al. 2017). In addition, NDCBE (the sodium-dependent chloride/bicarbonate exchanger, which works in association with pendrin to mediated the electroneutral Na+-Cl- absorption in ICs) is necessary for maintaining sodium balance and intravascular volume during salt depletion or NCC inactivation (Sinning, Radionov et al. 2017). Thus, all these studies are pointing to the importance of pendrin in the regulation of blood pressure in vivo; adding to the fact that intercalated cell salt reabsorptive pathway by NDCBE/Pendrin is thiazide-sensitive as NCC pathway. The thiazides drug, which main target is NCC, is one of the principal treatments for hypertensive patients. While low doses of thiazide are sufficient to block NCC activity (10 uM) (Sabath, Meade et al. 2004), the inhibition of NDCBE/Pendrin requires much higher doses (100 uM). Thus, when high doses of thiazide were used in patients (Greene, Escobar Quinones et al. 2007), the hypokalemia observed was severe, most likely because ENaC mediates the NaCl absorption in this case, consequently increasing the K+ excretion through ROMK. Accordingly, low doses of thiazide inhibit only NCC and compensatory activity of NDCBE/Pendrin may take place, lowering the rate of ENaC-dependent K+ secretion. In sum, this underlines the important contribution that NDCBE/Pendrin may have in PHA-like pathologies, not only in the Na+ balance but also indirectly in K+ handling. Moreover, our data indicate that pendrin overactivity is responsible for the bicarbonate leak observed in TgWNK4PHAII mice (Figure 15F), further suggesting that pendrin plays a role as a novel mechanism of renal tubular acidosis.

5.3 Impact of pendrin deletion on plasma K+ concentration of TgWnk4PHAII mice

Deletion of pendrin in TgWnk4PHAII mice caused only subtle changes in vascular volume, indicating that NCC hyperactivation is sufficient to drive hypertension in PHAII. However, genetic ablation of pendrin in PHAII mice fully corrects the hyperkalemia (Table 2); similarly to the findings that genetic ablation of NCC also alleviates hyperkalemia in TgWnk4PHAII mice (Lalioti, Zhang et al. 2006). On the contrary, isolated deletion of either NCC or pendrin in mouse has no or slight effect on blood K+ concentration under standard conditions of diet (Schultheis, Lorenz et al. 1998, Royaux, Wall et al. 2001, Verlander, Hassell et al. 2003, Wall, Kim et al. 2004), indicating that concomitant activation of NCC and pendrin/NDCBE pathways is necessary to drive hyperkalemia. Conversely, combined deletion of NCC and NDCBE induces hypokalemia (Sinning, Radionov et al. 2017). These findings indicate that these transport systems act in concert to control plasma K+ concentration.

Some studies have reported about the influence of plasma K+ level in the regulation of NCC and NDCBE/Pendrin pathway. Thus, hypokalemia activates NCC independently of aldosterone (Zhang, Wang et al. 2014, Terker, Zhang et al. 2015) while at the same time promotes pendrin induction through the aldosterone pathway. Therefore, controlling the progression of hypokalemia (Xu, Hirohama et al. 2017). Our result suggests that, in PHAII, these two mechanisms of prevention against hypokalemia become aberrantly activated and thus participate to the generation of hyperkalemia.

In TgWnk4PHAII mice, decreased ENaC-dependent K+ secretion was fully restored after deletion of pendrin (TgWnk4PHAII/Pds-/-), suggesting that K+ channels are affected by pendrin deregulation in PHAII. This is probably due to altered K+ channel activity since the natriuretic response to amiloride is normal and ROMK protein abundance unchanged (Figure 12). Similarly, in a WNK1-PHAII mouse model downregulation of ROMK and no change in ENaC were reported (Vidal- Petiot, Elvira-Matelot et al. 2013). Patch clamp experiments also report an inhibition of ROMK activity in the DCT2/early CNT of TgWnk4PHAII mice but, in this study, it is associated to a decrease in ENaC activity (Zhang, Wang et al. 2017). This is in contrast to another WKN4-PHAII model, where the authors found an

68 activation of ENaC (Yang, Morimoto et al. 2007). In sum, the pathogenesis of hyperkalemia seems to be independent of ENaC activity and expression.

The reversion of hyperkalemia by genetic ablation of pendrin in TgWnk4PHAII mice could be also due to enhanced BK activity in IC cells. BK4 subunit is present with BK subunit in IC cells as well as L-WNK1, which activates BK (Liu, Song et al. 2015). Chloride binding to WNK1 precludes its autophosphorylation and thereby inhibits kinase activity (Piala, Moon et al. 2014). It is thus possible that inactivation of pendrin by decreasing intracellular Cl- concentration activates L-WNK1 selectively in ICs, which in turns activates BK.

Additionally, potassium handling cannot be understood without taking in consideration the acid-base status. Thus, one could explain the hyperkalemia correction by deletion of pendrin as a secondary effect to the normalization of the acid-base status in TgWnk4PHAII mice. Changes in acid-base status affect potassium excretion in the distal nephron; specifically, metabolic acidosis inhibits whereas metabolic alkalosis stimulates K+ secretion (Aronson and Giebisch 2011). Consistent with this observation, it has been demonstrated that intracellular pH is an important modulator of the native ROMK channel in the CCD (Wang, Schwab et al. 1990, Wang 1995), such that acidification within the physiological range results in a reduction of single channel activity. Thus, correction of the acidosis following pendrin deletion would remove the acidic pH inhibition of ROMK activity.

Overall, many can be the hypothesis but the mechanisms by which genetic ablation of pendrin reverse hyperkalemia in TgWnk4PHAII mice are unknown and more studies are needed. So far, In vitro experiments have shown direct inhibitory effect of WNK4 on ROMK or BK (Kahle, Wilson et al. 2003, Zhuang, Zhang et al. 2011), contrary to our finding where ROMK protein abundance was not different between TgWnk4PHAII and control mice, moreover, an increase in α-BKCa abundance was observed in TgWnk4PHAII mice (Figure 13). Furthermore, an important point is to evaluate whether the deletion of pendrin in TgWnk4PHAII mice is still sufficient to keep in a normal range the plasmatic K+ concentration after challenging the double transgenic mice (TgWnk4PHAII/Pds-/-) with a high K+ diet.

5.4 Pendrin hyperactivity as a novel mechanism for renal acidosis

One of the most exciting findings of our study is the identification of pendrin hyperactivity as a novel mechanism for renal acidosis. Certainly, our experiments shown in figures 14 and 15 clearly demonstrated that the metabolic acidosis is not caused by hyperkalemia or by defective V-ATPase activity. Indeed, 1) metabolic acidosis was not significantly alleviated by the normalization of blood K+ concentration; 2) ammonium excretion and proton secretion were not impaired and the mice were still able to achieve maximal urine acidification and ammonium excretion under stimulated condition. By contrast, our results showed that pendrin- - - mediated Cl /HCO3 exchange per cell as well as the total number of pendrin expressing cells, are both markedly increased, while pendrin genetic ablation corrects completely metabolic acidosis in TgWnk4PHAII mice indicate that metabolic acidosis is caused by excessive bicarbonate secretion in the distal nephron. Activation of pendrin in TgWnk4PHAII mice is accompanied by an alteration of the structure of the CCD. Indeed, we show an increase in -intercalated cells at the expense of -intercalated cells in TgWnk4PHAII mice. Several studies report that acidosis induces the differentiation of β-ICs into α-ICs (Al-Awqati and Gao 2011) but only one study reports IC conversion in the opposite direction. This is in a SPAK knock out mouse model that expresses NCC at a very low level (McCormick, Mutig et al. 2011). An increased number of pendrin positive-IC cells was also observed in NCC knock out mice as the result of hyperplasia of the CNT (Loffing, Vallon et al. 2004, Vallet, Picard et al. 2006).

Interestingly, if the metabolic acidosis is caused by a distal renal leak of bicarbonate, it may seem puzzling that TgWnk4PHAII mice did not exhibit obvious bicarbonaturia. However, this is also the case in patients with proximal renal acidosis when they are in steady state, i.e., when they have overt metabolic acidosis, due to compensatory mechanisms (Rodriguez Soriano, Boichis et al. 1967, Soriano, Boichis et al. 1967, Rodriguez Soriano 2002, Haque, Ariceta et al. 2012). Otherwise, these patients would not be in steady state and would die from uncontrolled acidosis.

Remarkably, it has been recently demonstrated that calcineurin inhibitors, which are extensively used as immunosuppressive agents in organ transplantation, alter

70 the WNK signaling pathway, and thereby, can activate NCC and cause hypertension (Hoorn, Walsh et al. 2011). These patients do not exhibit only hypertension but are also frequently affected by hyperkalemia and metabolic acidosis (Keven, Ozturk et al. 2007, Tanrisev, Gungor et al. 2015). It is tempting to speculate that pendrin hyperactivity will be also detected in these patients and can account for these symptoms.

In conclusion, our study demonstrates a central role of pendrin in acid-base and potassium homeostasis, and identifies a renal leak of bicarbonate by the distal nephron as a novel type of renal tubular acidosis.

5.5 The missing link between pendrin regulation and WNK4

Our results show that thiazide-sensitive electroneutral NaCl absorption through the pendrin/NDCBE transport system is activated in isolated CCDs of TgWnk4PHAII mice (Figure 10 and 18). The fact that WNK4 is expressed not only in the DCT cells but also in intercalated cells of the CCD (Wilson, Disse-Nicodeme et al. 2001, Ohno, Uchida et al. 2011, Shibata, Rinehart et al. 2013), suggests a link between WNK4 and pendrin regulation. Many hypotheses have been put forward. One example is the identification of a new phosphorylation site (S843) in the mineralocorticoid receptor (MRS843-P), which is phosphorylated exclusively in IC and prevents ligand binding. ANG II and WNK4 signaling decrease MRS843-P levels, thereby restoring MR activation by aldosterone and increasing the expression of pendrin and the proton pump in ICs. These results support the role of an ANG II/WNK4 pathway in the regulation of the vH+-ATPase-dependent NDCBE/pendrin NaCl transport system (Shibata, Rinehart et al. 2013). Consistently, TgWnk4PHAII mice (gain of function of WNK4) have lower levels of MRS843-P while Wnk4 knockout mice showed increased levels of MRS843-P.

Interestingly Na+ absorption in CCDs from Wnk4 knock-out mice (in which NCC was dramatically downregulated) does not occur through the pendrin/NDCBE system but through ENaC (Castaneda-Bueno, Cervantes-Perez et al. 2012). This model differs from Ncc knock-out mice, where pendrin-dependent NaCl transport was the dominant mechanism accounting for Na+ absorption in the CCD (Leviel,

Hubner et al. 2010). Moreover, in Ncc knock-out mice levels of MRS843-P were dramatically decreased (Shibata, Rinehart et al. 2013).

Taken together, these studies strongly suggest that WNK4 regulates pendrin, but in vitro experiments must be performed in order to have the final proof. For instance, to demonstrate that increase in pendrin activity in -ICs results directly from the expression of the WNK4 mutant in these cells, one could perform an experiment with the co-injection in xenopus oocytes of pendrin cRNA with a WNK4 cRNA bearing the same PHAII-mutation as the one used to generate the TgWNK4PHAII animals (WNK4-Q562E). Thus, the effect of WNK4 and WNK4 mutant on pendrin activity can be determined by measuring chloride transport (36Cl− influx). Similar experiment could be performed to evaluate the impact of WNK4 mutant on NDCBE, considering that pendrin work in conjugation with NDCBE. Furthermore, the generation of a double transgenic mouse model by the genetic deletion of NDCBE in TgWNK4PHAII mice may highly contribute to our conclusion. Also compare our results with the deletion of pendrin in others PHAII mice models could help to assess the extent of the pendrin in the pathogenesis of PHAII.

5.6 Conclusion

The mice models have emerged as a possible solution for overcoming the many time, contradictory results derived from in vitro experiments. Several PHAII mice models have been generated with this aim in view but the outcome appears mitigated: the increase of NCC abundance and activity is certainly common to all models but contradictory data have been obtained for ENaC and ROMK, which are also main players in NaCl and K+ transport.

We propose pendrin as a new actor in the pathogenesis of PHAII. We suggest that the metabolic acidosis observed in TgWNK4PHAII mice is a consequence of the increase in pendrin activity, which leads to a renal bicarbonate leak. Moreover, genetic ablation of pendrin in TgWNK4PHAII mice, also alleviate the hyperkalemia by a mechanism that must be further investigated. In this line, it is important to mention that a mouse model overexpressing pendrin in ICs does not display a PHAII-like phenotype in normal salt diet but only develops hypertension when

72 exposed to a high-salt diet. Moreover, the increased electroneutral NaCl absorption in the distal nephron does not lead to high serum K+ concentration or metabolic acidosis (Jacques, Picard et al. 2013). All together, these data suggest that pendrin overactivity is not sufficient to cause the whole PHAII phenotype and likely needs to be associated with NCC upregulation, both contributing to the pathogenesis of PHAII. Thus, further investigation is needed to fully understand the contribution of pendrin in the pathophysiology of this syndrome. The fact that CNT is a nephron segment not suitable for microperfusion experiment, is still representing a problem, since ENaC, pendrin and ROMK are also expressed in this nephron segment and their contribution to the onset of PHAII, particularly there, is still unkown.

Elucidate the pathogenesis of PHAII and more generally increase our knowledge of blood pressure related diseases, is important since only half of hypertensive patients currently exhibit an appropriate control of their blood pressure despite the existence of different and complementary classes of antihypertensive therapies. Most patients (>75%) require a combination of at least two antihypertensive drugs and others require up to five different compounds. The actual effectiveness of some antihypertensive treatments to decrease blood pressure, and more importantly, to increase the lifespan of the patients is still a matter of discussion. Thus, the development of new drugs targeting blood pressure is clearly required to establish effective therapies for hypertension. Therefore, the fact that primary changes in chloride transport can drive NaCl absorption has important therapeutic implications, our results suggest that pendrin might be a very interesting target to modulate NaCl absorption by the distal nephron; pendrin blockers could, consequently, represent a new class of antihypertensive compounds.

6. PUBLICATIONS

The paper for the presented thesis, was submitted to Kidney international

A Mouse Model of Pseudohypoaldosteronism type II Reveals a Novel Mechanism of Renal Tubular Acidosis

Karen I. López-Cayuqueo1,2*, Maria Chavez-Canales1*, Alexia Pillot3, Pascal Houillier3,4, 1 6 1,† 1 Maximilien Jayat , Jennifer Baraka-Vidot , Francesco Trepiccione , Véronique Baudrie , Cara Büsst1$, Christelle Soukaseum1, Yusuke Kumai1, Xavier Jeunemaître1,4, Juliette Hadchouel1, Dominique Eladari5, 6, §, Régine Chambrey1, 6,7, § 1INSERM U970, Paris Cardiovascular Research Center; Université Paris-Descartes, 56 rue Leblanc, F-75015 Paris, France 2Centro de Estudios Científicos (CECs), Valdivia, Chile 3Centre National de la Recherche Scientifique Equipe de Recherche Labelisée 8228, Institut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche_S1138, Centre de Recherche des Cordeliers, Université Pierre et Marie Curie, Paris, France. 4Faculté de Médecine, Université Paris-Descartes, Paris, France; Département de Génétique, Hôpital Européen Georges Pompidou, Assistance Publique-Hôpitaux de Paris, Paris, France 5Service d'Explorations Fonctionnelles Rénales, Hôpital Felix Guyon, CHU de la Réunion, St Denis F-97400, Ile de la Réunion, France 6Inserm U1188, Diabète athérothrombose Thérapies Réunion Océan Indien (DéTROI) ; Université de La Réunion ; CYROI, 2, rue Maxime Rivière, 97490 Sainte Clotilde, La Réunion, France 7Centre National de la Recherche Scientifique, Délégation Paris Michel-Ange, France

* Karen I. López-Cayuqueo and Maria Chavez share first authorship. § R.Chambrey and D. Eladari share senior authorship.

Corresponding author: Dr Régine Chambrey, Inserm U1188, CYROI, 2, rue Maxime Rivière, 97490 Sainte Clotilde, La Réunion, France phone : +262 6 92 15 42 79 e-mail: [email protected] or Dr Dominique Eladari, Service d'Explorations Fonctionnelles Rénales, Hôpital Felix Guyon, CHU de la Réunion, St Denis F-97400, Ile de la Réunion, France Phone: +262 6 92 16 38 48 e-mail: [email protected]

Moreover, in my time in the laboratory of Dr. Dominique Eladari, at the Paris Cardiovascular Research Center (PARCC), as part of my doctoral formation I contributed in some projects, which results were published in the following articles:

 Masayoshi Nanami, Truyen D. Pham1, Young Hee Kim, Baoli Yang, Roy L. Sutliff, Olivier Staub, Janet D. Klein, Karen I. Lopez-Cayuqueo, Regine Chambrey, Annie Y. Park, Xiaonan Wang, Vladimir Pech, Jill W. Verlander and Susan M. Wall. Intercalated Cell Nedd4-2: Role in blood pressure regulation, ion transport and transporter expression. The Journal of Physiology, under revision.

 J Christopher Hennings, Olga Andrini, Nicolas Picard, Marc Paulais, Antje K Huebner, Irma Karen Lopez Cayuqueo, Yohan Bignon, Mathilde Keck, Nicolas Cornière, D. Bohm, Thomas J Jentsch, Régine Chambrey, Jacques Teulon, C. A. Hubner, Dominique Eladari: The ClC-K2 Chloride Channel Is Critical for Salt Handling in the Distal Nephron. Journal of the American Society of Nephrology 06/2016; DOI:10.1681/ASN.2016010085  Anne Sinning, Nikita Radionov, Francesco Trepiccione, Karen I López- Cayuqueo, Maximilien Jayat, Stéphanie Baron, Nicolas Cornière, R Todd Alexander, Juliette Hadchouel, Dominique Eladari, C. A. Hubner, Régine Chambrey: Double Knockout of the Na+-Driven Cl-/HCO3- Exchanger and Na+/Cl- Cotransporter Induces Hypokalemia and Volume Depletion. Journal of the American Society of Nephrology 05/2016; DOI:10.1681/ASN.2015070734  Francesco Trepiccione, Simon D Gerber, Florian Grahammer, Karen I López-Cayuqueo, Véronique Baudrie, T. G. Paunescu, Diane E Capen, Nicolas Picard, R Todd Alexander, Tobias B Huber, Regine Chambrey, Dennis Brown, Pascal Houillier, Dominique Eladari, Matias Simons: Renal Atp6ap2/(Pro)renin Receptor Is Required for Normal Vacuolar H+-ATPase Function but Not for the Renin-Angiotensin System. Journal of the American Society of Nephrology 04/2016; DOI:10.1681/ASN.2015080915  Mohammed Z. Ferdaus, Karl W. Barber, Karen I. López-Cayuqueo, Andrew S. Terker, Eduardo R. Argaiz, Brandon M. Gassaway, Régine Chambrey, Gerardo Gamba, Jesse Rinehart, James A. McCormick: SPAK

and OSR1 play essential roles in potassium homeostasis through actions on the distal convoluted tubule. The Journal of Physiology 04/2016; DOI:10.1113/JP272311

Furthermore, my PhD was related to the Chilean Laboratory of Dr. Francisco Sepulveda from Centro de Estudios Científicos (CECs), where my results contributed to the following 2 papers:

 Isabel Cornejo, Johanna Burgos, Sandra Villanueva, Karen López- Cayuqueo, Régine Chambrey, Neudo Buelvas, María Isabel Niemeyer, Dulce Figueiras-Fierro, Peter D. Brown, Francisco V. Sepúlveda & L. Pablo Cid. Tissue distribution of Kir7.1 inwardly rectifying K+ channel probed in a knockin mouse expressing a haemagglutinin-tagged protein. Frontiers in Physiology, under revision.  Sandra Villanueva, Johanna Burgos, Karen I López-Cayuqueo, Ka-Man Venus Lai, David M Valenzuela, L Pablo Cid, Francisco V Sepúlveda: Cleft Palate, Moderate Lung Developmental Retardation and Early Postnatal Lethality in Mice Deficient in the Kir7.1 Inwardly Rectifying K+ Channel. PLoS ONE 09/2015; 10(9). DOI:10.1371/journal.pone.0139284

Additional publications:

 Karen I López-Cayuqueo, Gaspar Peña-Münzenmayer, María Isabel

+ Niemeyer, Francisco V Sepúlveda, I. Pablo Cid: TASK-2 K2P K channel: Thoughts about gating and its fitness to physiological function. Pflügers Archiv - European Journal of Physiology 10/2014; 467(5). DOI:10.1007/s00424-014-1627-7

7. ACKNOWLEDGMENTS

I would like to thank Prof. Pierre Ronco, Prof. Renaud Beauwens, Dr. Henrik Dimke and Dr. Maria Christina Zennaro, to kindly accept to be part of the committee of my thesis.

My gratitude to Dr. Régine Chambrey and Dr. Dominique Eladari for giving me the opportunity to work together.

I thank to Prof. Jacques Teulon for being a huge support during all my PhD. I also would like to thank Dr. Francisco Sepulveda and Prof. Pablo Cid, members of my former laboratory, Centro de Estudios Científicos (CECs), from Valdivia, Chile.

I want to thank to the members of the laboratory of Prof. Thomas Jentsch, where I spent the last part of my doctoral formation.

Finally special thanks to my family: Parents and brother and to my mapuches grandparents.

For performing my PhD I had the support of the Chilean government through ‘’Becas Chile-Doctorado en el extranjero” fellowship program from CONICYT.

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