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BackgroundNephrolithiasis (NL) is a complex multifactorial disease affecting up to 10%–20% of the human population and causing a significant burden on public health systems worldwide. It results from a combination of environmental and genetic factors. Hyperoxaluria is a major risk factor for NL.MethodsWe used a whole exome-based approach in a patient with calcium oxalate NL. The effects of the mutation were characterised using cell culture and in silico analyses.ResultsWe identified a rare heterozygous missense mutation (c.1519C>T/p.R507W) in theSLC26A6gene that encodes a secretory oxalate transporter. This mutation cosegregated with hyperoxaluria in the family. In vitro characterisation of mutant SLC26A6 demonstrated that Cl−-dependent oxalate transport was dramatically reduced because the mutation affects both SLC26A6 transport activity and membrane surface expression. Cotransfection studies demonstrated strong dominant-negative effects of the mutant on the wild-type protein indicating that the phenotype of patients heterozygous for this mutation may be more severe than predicted by haploinsufficiency alone.ConclusionOur study is in line with previous observations made in the mouse showing thatSLC26A6inactivation can cause inherited enteric hyperoxaluria with calcium oxalate NL. Consistent with an enteric form of hyperoxaluria, we observed a beneficial effect of increasing calcium in the patient’s diet to reduce urinary oxalate excretion.

BackgroundNephrotic syndrome (NS) is characterized by massive sodium chloride retention. Along the kidney tubule, sodium and chloride reabsorption are coupled via a combination of transcellular and paracellular transport pathways. The mechanism of sodium retention in NS has been extensively studied, but the associated chloride transport pathway has not been elucidated.MethodsTo investigate the pathway of chloride retention in NS, we assessed the expression levels of both paracellular and transcellular components of chloride transport in the CD of POD-ATTAC mice and PAN rats, two rodent models of NS. We also used cultured mouse cortical collecting duct cells to see how overexpression or silencing of claudin-4 affect paracellular permeability. Finally, human renal biopsies were used to confirm our in vivo results.ResultsIn control animals, claudin-4 was expressed at low levels in collecting duct (CD). In POD-ATTAC mice and PAN rats, claudin-4 expression was strongly increased in CD beta-intercalated cells (B-IC) and to a lesser extent in CD principal cells and was also induced in connecting tubules. Similarly, we found that claudin-4 was expressed at low levels in normal human kidneys and was dramatically increased in CD cells of nephrotic human kidneys (focal and segmental glomerulosclerosis). In parallel, the expression of pendrin, which exchanges chloride for bicarbonates in B-IC, was decreased in nephrotic compared to control animals. However, the increase in claudin-4 expression observed in NS is likely independent of pendrin abundance. Increased claudin-4 abundance is coupled with increased ENaC-dependent sodium transport. Overexpression or silencing of claudin-4 in mCCDcl1 cells confirmed the preferential permeability of claudin-4 to chloride over sodium.ConclusionsThese results suggest that during NS, transcellular Cl-/HCO - transport decreases while paracellular chloride transport via claudin-4 may increase along the collecting system. Paracellular chloride permeability may constitute a chloride shunt that favors Na+ reabsorption and opposes K+ secretion along the CD in NS.Significance StatementNephrotic syndrome is a common disease characterized by massive proteinuria, hypoalbuminemia and edema due to renal sodium-chloride retention. We demonstrate for the first time an induction of claudin-4 expression indicating a partial shift from transcellular to paracellular chloride transport in the renal collecting system of nephrotic rodents. We confirmed the increased expression of claudin-4 in kidney biopsies of nephrotic patients, highlighting the translational significance of these results. Whether the paracellular pathway may represent a novel target to treat edema in nephrotic syndrome remains to be elucidated.

Distal renal tubular acidosis is a rare renal tubular disorder characterized by hyperchloremic metabolic acidosis and impaired urinary acidification. Mutations in three genes (ATP6V0A4, ATP6V1B1 and SLC4A1) constitute a monogenic causation in 58–70% of familial cases of distal renal tubular acidosis. Recently, mutations in FOXI1 have been identified as an additional cause. Therefore, we hypothesized that further monogenic causes of distal renal tubular acidosis remain to be discovered. Panel sequencing and/or whole exome sequencing was performed in a cohort of 17 families with 19 affected individuals with pediatric onset distal renal tubular acidosis. A causative mutation was detected in one of the three “classical” known distal renal tubular acidosis genes in 10 of 17 families. The seven unsolved families were then subjected to candidate whole exome sequencing analysis. Potential disease causing mutations in three genes were detected: ATP6V1C2, which encodes another kidney specific subunit of the V-type proton ATPase (1 family); WDR72 (2 families), previously implicated in V-ATPase trafficking in cells; and SLC4A2 (1 family), a paralog of the known distal renal tubular acidosis gene SLC4A1. Two of these mutations were assessed for deleteriousness through functional studies. Yeast growth assays for ATP6V1C2 revealed loss-of-function for the patient mutation, strongly supporting ATP6V1C2 as a novel distal renal tubular acidosis gene. Thus, we provided a molecular diagnosis in a known distal renal tubular acidosis gene in 10 of 17 families (59%) with this disease, identified mutations in ATP6V1C2 as a novel human candidate gene, and provided further evidence for phenotypic expansion in WDR72 mutations from amelogenesis imperfecta to distal renal tubular acidosis.

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

Background: Distal renal tubular acidosis (dRTA) is characterized by an impairment of the urinary acidification process in the distal nephron. Complete or incomplete metabolic acidosis coupled with inappropriately alkaline urine are the hallmarks of this condition. Genetic forms of dRTA are caused by loss of function mutations of either SLC4A1, encoding the AE1 anion exchanger, or ATP6V1B1 and ATP6V0A4, encoding for the B1 and a4 subunits of the vH+ATPase, respectively. These genes are crucial for the function of A-type intercalated cells (A-IC) of the distal nephron. Summary: Alterations of acid-base homeostasis are variably associated with hypokalemia, hypercalciuria, nephrocalcinosis or nephrolithiasis, and a salt-losing phenotype. Here we report the diagnostic test and the underlying physiopathological mechanisms. The molecular mechanisms identified so far can explain the defect in acid secretion, but do not explain all clinical features. We review the latest experimental findings on the pathogenesis of dRTA, reporting mechanisms that are instrumental for the clinician and potentially inspiring a novel therapeutic strategy. Key Message: Primary dRTA is usually intended as a single-cell disease because the A-IC are mainly affected. However, novel evidence shows that different cell types of the nephron may contribute to the signs and symptoms, moving the focus from a single-cell towards a renal disease.

Objectif Le syndrome d’apnees du sommeil (SAS) est une pathologie frequente mais sous-estimee. Les malades renaux chroniques (MRC) ne sont actuellement pas cites dans les recommandations francaises, comme un groupe a risque de SAS. Nous estimons, dans notre etude, la prevalence du SAS et sa severite dans une cohorte de MRC du CHU de La Reunion. Methodes Il s’agit d’une etude observationnelle retrospective portant sur une population de malades renaux chroniques de stades 1 a 4, definis par une mesure du debit de filtration glomerulaire (DFG) a l’EDTA, ayant accepte un enregistrement du sommeil par polygraphie ventilatoire. Le SAS etait defini par un IAH > 5/H. Resultats Cent trente-trois patients malades renaux chroniques ont ete inclus. Un SAS de tout stade confondu a ete retrouve chez 51 % des patients (n = 68), majoritairement obstructif (n = 60) dont 47 patients (35 %) presentant un SAS modere a severe (IAH > 15/H). Le sexe masculin et l’âge etaient significativement associes au diagnostic de SAS. Le DFG etait plus bas dans le groupe SAS que dans le groupe non SAS (47,8 ± 19,6 vs 59 ± 21,16 mL/min/1,73 m2, p Conclusion Nous avons retrouve une forte prevalence du SAS dans notre population. Une etude prospective permettrait de mieux caracteriser les facteurs de risque qui y sont associes chez les malades renaux chroniques, et ses consequences sur le DFG et son declin.

The distal nephron is a heterogeneous part of the nephron composed by six different cell types, forming the epithelium of the distal convoluted (DCT), connecting, and collecting duct. To dissect the function of these cells, knockout models specific for their unique cell marker have been created. However, since this part of the nephron develops at the border between the ureteric bud and the metanephric mesenchyme, the specificity of the single cell markers has been recently questioned. Here, by mapping the fate of the aquaporin 2 (AQP2) and Na+-Cl−cotransporter (NCC)-positive cells using transgenic mouse lines expressing the yellow fluorescent protein fluorescent marker, we showed that the origin of the distal nephron is extremely composite. Indeed, AQP2-expressing precursor results give rise not only to the principal cells, but also to some of the A- and B-type intercalated cells and even to cells of the DCT. On the other hand, some principal cells and B-type intercalated cells can develop from NCC-expressing precursors. In conclusion, these results demonstrate that the origin of different cell types in the distal nephron is not as clearly defined as originally thought. Importantly, they highlight the fact that knocking out a gene encoding for a selective functional marker in the adult does not guarantee cell specificity during the overall kidney development. Tools allowing not only cell-specific but also time-controlled recombination will be useful in this sense.

The value of estimated glomerular filtration rate (eGFR) in living kidney donors screening is unclear. A recently published web-based application derived from large cohorts, but not living donors, calculates the probability of a measured GFR (mGFR) lower than a determined threshold. Our objectives were to validate the clinical utility of this tool in a cohort of living donors and to test two other strategies based on chronic kidney disease epidemiology collaboration (CKD-EPI) and on MDRD-eGFR. GFR was measured using 51 Cr- ethylene-diamine tetraacetic acid urinary clearance in 311 potential living kidney donors (178 women, mean age 50 ± 11.6 years). The web-based tool was used to predict those with mGFR < 80 mL/min/1.73 m2 . Inputs to the application were sex, age, ethnicity, and plasma creatinine. In our cohort, a web-based probability of mGFR

Background and objectives The predictors of long–term renal function in living kidney donors are currently discussed. Our objectives were to describe the predictors of functional gain of the remaining kidney after kidney donation. We hypothesized that GFR of the remaining kidney divided by volume of this kidney (rk-GFR/vol) would reflect the density of functional nephrons and be inversely associated with functional gain of the remaining kidney. Design, setting, participants, & measurements We conducted a prospective monocentric study including 63 living donors (26 men; 50.3±11.8 years old) who had been evaluated for 51Cr-EDTA and measured GFR, split renal function by scintigraphy before donation (between 2004 and 2009), and measured GFR at 5.7±0.5 years after donation. For 52 donors, volume of the remaining kidney (measured and estimated with the ellipsoid formula using renal computed tomography scannography) was determined before donation. We tested our hypothesis in an external validation cohort of 39 living donors (13 men; 51.0±9.4 years old) from another single center during the same time period. Results For the main cohort, the mean measured GFR was 97.6±13.0 ml/min per 1.73 m2 before donation and 63.8±9.4 ml/min per 1.73 m2 at 5 years. Functional gain averaged 16.2±7.2 ml/min per 1.73 m2 (+35.3%±16.7%). Multivariate analysis showed that age, body mass index, and rk-GFR/vol at donation were negatively correlated with functional gain and had strong predictive power of the 5-year functional gain (adjusted 5-year functional gain for age: −0.4 [95% confidence interval (95% CI), −0.5 to −0.1]; body mass index: −0.3 [95% CI, −0.6 to −0.1]; rk-GFR/vol: −55.1 [95% CI, −92.3 to −17.9]). We tested this model in the external validation cohort (adjusted 5-year functional gain for age: −0.1 [95% CI, −0.5 to 0.3]; body mass index: −0.9 [95% CI, −1.8 to −0.1]; rk-GFR/vol: −97.6 [95% CI, −137.5 to −57.6]) and confirmed that rk-GFR/vol was inversely associated with 5-year functional gain. Conclusions For given age and body mass index, the long–term functional gain of the remaining kidney is inversely associated with the new variable rk-GFR/vol at donation.

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

The nephron cortical collecting duct (CCD) is composed of principal cells, which mediate Na, K, and water transport, and intercalated cells (ICs), which are specialized for acid-base transport. There are two canonical IC forms: acid-secreting α-ICs and HCO3-secreting β-ICs. Chronic acidosis increases α-ICs at the expense of β-ICs, thereby increasing net acid secretion by the CCD. We found by growth factor quantitative PCR array that acidosis increases expression of mRNA encoding SDF1 (or CXCL12) in kidney cortex and isolated CCDs from mouse and rabbit kidney cortex. Exogenous SDF1 or pH 6.8 media increased H+ secretion and decreased HCO3 secretion in isolated perfused rabbit CCDs. Acid-dependent changes in H+ and HCO3 secretion were largely blunted by AMD3100, which selectively blocks the SDF1 receptor CXCR4. In mice, diet-induced chronic acidosis increased α-ICs and decreased β-ICs. Additionally, IC-specific Cxcr4 deletion prevented IC subtype alterations and magnified metabolic acidosis. SDF1 was transcriptionally regulated and a target of the hypoxia-sensing transcription factor HIF1α. IC-specific deletion of Hif1a produced no effect on mice fed an acid diet, as α-ICs increased and β-ICs decreased similarly to that observed in WT littermates. However, Hif1a deletion in all CCD cells prevented acidosis-induced IC subtype distribution, resulting in more severe acidosis. Cultured principal cells exhibited an HIF1α-dependent increase of Sdf1 transcription in response to media acidification. Thus, our results indicate that principal cells respond to acid by producing SDF1, which then acts on adjacent ICs.

Because majority of biological processes are dependent on pH, maintaining systemic acid-base balance is critical. The kidney contributes to systemic acid-base regulation, by reabsorbing HCO3 (-) (both filtered by glomeruli and generated within a nephron) and acidifying urine. Abnormalities in those processes will eventually lead to a disruption in systemic acid-base balance and provoke metabolic acid-base disorders. Research over the past 30 years advanced our understanding on cellular and molecular mechanisms responsible for those processes. In particular, a variety of transgenic animal models, where target genes are deleted either globally or conditionally, provided significant insights into how specific transporters are contributing to the renal acid-base regulation. Here, we broadly overview the mechanisms of renal ion transport participating to acid-base regulation, with emphasis on data obtained from transgenic mice models.

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

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

International audience; Background: Pendrin, the chloride/bicarbonate exchanger of β-intercalated cells of the renal connecting tubule and the collecting duct, plays a key role in NaCl reabsorption by the distal nephron. Therefore, pendrin may be important for the control of extracellular fluid volume and blood pressure.Methods: Here, we have used a genetic mouse model in which the expression of pendrin can be switched-on in vivo by the administration of doxycycline. Pendrin can also be rapidly removed when doxycycline administration is discontinued. Therefore, our genetic strategy allows us to test selectively the acute effects of loss of pendrin function.Results: We show that acute loss of pendrin leads to a significant decrease of blood pressure. In addition, acute ablation of pendrin did not alter significantly the acid-base status or blood K + concentration.Conclusion: By using a transgenic mouse model, avoiding off-target effects related to pharmacological compounds, this study suggests that pendrin could be a novel target to treat hypertension.

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

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

Metabolic acidosis is associated with increased urinary calcium excretion and related sequelae, including nephrocalcinosis and nephrolithiasis. The increased urinary calcium excretion induced by metabolic acidosis predominantly results from increased mobilization of calcium out of bone and inhibition of calcium transport processes within the renal tubule. The mechanisms whereby acid alters the integrity and stability of bone have been examined extensively in the published literature. Here, after briefly reviewing this literature, we consider the effects of acid on calcium transport in the renal tubule and then discuss why not all gene defects that cause renal tubular acidosis are associated with hypercalciuria and nephrocalcinosis.

Inactivation of the B1 proton pump subunit (ATP6V1B1) in intercalated cells (ICs) leads to type I distal renal tubular acidosis (dRTA), a disease associated with salt- and potassium-losing nephropathy. Here we show that mice deficient in ATP6V1B1 (Atp6v1b1-/- mice) displayed renal loss of NaCl, K+, and water, causing hypovolemia, hypokalemia, and polyuria. We demonstrated that NaCl loss originated from the cortical collecting duct, where activity of both the epithelial sodium channel (ENaC) and the pendrin/Na(+)-driven chloride/bicarbonate exchanger (pendrin/NDCBE) transport system was impaired. ENaC was appropriately increased in the medullary collecting duct, suggesting a localized inhibition in the cortex. We detected high urinary prostaglandin E2 (PGE2) and ATP levels in Atp6v1b1-/- mice. Inhibition of PGE2 synthesis in vivo restored ENaC protein levels specifically in the cortex. It also normalized protein levels of the large conductance calcium-activated potassium channel and the water channel aquaporin 2, and improved polyuria and hypokalemia in mutant mice. Furthermore, pharmacological inactivation of the proton pump in β-ICs induced release of PGE2 through activation of calcium-coupled purinergic receptors. In the present study, we identified ATP-triggered PGE2 paracrine signaling originating from β-ICs as a mechanism in the development of the hydroelectrolytic imbalance associated with dRTA. Our data indicate that in addition to principal cells, ICs are also critical in maintaining sodium balance and, hence, normal vascular volume and blood pressure.

Activation of the intrarenal renin-angiotensin system (RAS) can elicit hypertension independently from the systemic RAS. However, the precise mechanisms by which intrarenal Ang II increases blood pressure have never been identified. To this end, we studied the responses of mice specifically lacking kidney angiotensin-converting enzyme (ACE) to experimental hypertension. Here, we show that the absence of kidney ACE substantially blunts the hypertension induced by Ang II infusion (a model of high serum Ang II) or by nitric oxide synthesis inhibition (a model of low serum Ang II). Moreover, the renal responses to high serum Ang II observed in wild-type mice, including intrarenal Ang II accumulation, sodium and water retention, and activation of ion transporters in the loop of Henle (NKCC2) and distal nephron (NCC, ENaC, and pendrin) as well as the transporter activating kinases SPAK and OSR1, were effectively prevented in mice that lack kidney ACE. These findings demonstrate that ACE metabolism plays a fundamental role in the responses of the kidney to hypertensive stimuli. In particular, renal ACE activity is required to increase local Ang II, to stimulate sodium transport in loop of Henle and the distal nephron, and to induce hypertension.

Two end-stage renal disease (ESRD) risk calculators were recently developed by Grams et al., and Ibrahim et al. to calculate ESRD risk before donation among living kidney donors. However, those calculators have never been studied among potential donors for whom donation was refused due to medical contraindications and compared to a group of donors. We compared 15-year and lifetime ESRD risk of donors and nondonors due to medical cause as estimated by those two calculators. Nondonors due to medical cause (n = 27) had a significantly higher 15-year ESRD risk compared to donors (n = 288) with both calculators (0.25 vs. 0.14, P < 0.001 for that developed by Grams et al. and 2.21 vs. 1.43, P = 0.002 for that developed by Ibrahim et al.). On the contrary, lifetime ESRD risk was not significantly different between the two groups. At both times (15 years and lifetime), we observed a significant overlap of ESRD risk between the two groups. ESRD risk calculators could be complementary to standard screening strategy but cannot be used alone to accept or decline donation.

Distal nephron acid secretion is mediated by highly specialized type A intercalated cells (A-ICs), which contain vacuolar H+-ATPase (V-type ATPase)-rich vesicles that fuse with the apical plasma membrane on demand. Intracellular bicarbonate generated by luminal H+ secretion is removed by the basolateral anion-exchanger AE1. Chronically reduced renal acid excretion in distal renal tubular acidosis (dRTA) may lead to nephrocalcinosis and renal failure. Studies in MDCK monolayers led to the proposal of a dominant-negative trafficking mechanism to explain AE1-associated dominant dRTA. To test this hypothesis in vivo, we generated an Ae1 R607H knockin mouse, which corresponds to the most common dominant dRTA mutation in human AE1, R589H. Compared with wild-type mice, heterozygous and homozygous R607H knockin mice displayed incomplete dRTA characterized by compensatory upregulation of the Na+/HCO3− cotransporter NBCn1. Red blood cell Ae1-mediated anion-exchange activity and surface polypeptide expression did not change. Mutant mice expressed far less Ae1 in A-ICs, but basolateral targeting of the mutant protein was preserved. Notably, mutant mice also exhibited reduced expression of V-type ATPase and compromised targeting of this proton pump to the plasma membrane upon acid challenge. Accumulation of p62- and ubiquitin-positive material in A-ICs of knockin mice suggested a defect in the degradative pathway, which may explain the observed loss of A-ICs. R607H knockin did not affect type B intercalated cells. We propose that reduced basolateral anion-exchange activity in A-ICs inhibits trafficking and regulation of V-type ATPase, compromising luminal H+ secretion and possibly lysosomal acidification.

International audience; ATPase H+-transporting lysosomal accessory protein 2 (Atp6ap2), also known as the (pro)renin receptor, is a type 1 transmembrane protein and an accessory subunit of the vacuolar H+-ATPase (V-ATPase) that may also function within the renin-angiotensin system. However, the contribution of Atp6ap2 to renin-angiotensin-dependent functions remains unconfirmed. Using mice with an inducible conditional deletion of Atp6ap2 in mouse renal epithelial cells, we found that decreased V-ATPase expression and activity in the intercalated cells of the collecting duct impaired acid-base regulation by the kidney. In addition, these mice suffered from marked polyuria resistant to desmopressin administration. Immunoblotting revealed downregulation of the medullary Na+-K+-2Cl- cotransporter NKCC2 in these mice compared with wild-type mice, an effect accompanied by a hypotonic medullary interstitium and impaired countercurrent multiplication. This phenotype correlated with strong autophagic defects in epithelial cells of medullary tubules. Notably, cells with high accumulation of the autophagosomal substrate p62 displayed the strongest reduction of NKCC2 expression. Finally, nephron-specific Atp6ap2 depletion did not affect angiotensin II production, angiotensin II-dependent BP regulation, or sodium handling in the kidney. Taken together, our results show that nephron-specific deletion of Atp6ap2 does not affect the renin-angiotensin system but causes a combination of renal concentration defects and distal renal tubular acidosis as a result of impaired V-ATPase activity.

The V-ATPase is a multisubunit complex that transports protons across membranes. Mutations of its B1 or a4 subunit are associated with distal renal tubular acidosis and deafness. In the kidney, the a4 subunit is expressed in intercalated cells of the distal nephron, where the V-ATPase controls acid/base secretion, and in proximal tubule cells, where its role is less clear. Here, we report that a4 KO mice suffer not only from severe acidosis but also from proximal tubule dysfunction with defective endocytic trafficking, proteinuria, phosphaturia and accumulation of lysosomal material and we provide evidence that these findings may be also relevant in patients. In the inner ear, the a4 subunit co-localized with pendrin at the apical side of epithelial cells lining the endolymphatic sac. As a4 KO mice were profoundly deaf and displayed enlarged endolymphatic fluid compartments mirroring the alterations in pendrin KO mice, we propose that pendrin and the proton pump co-operate in endolymph homeostasis. Thus, our mouse model gives new insights into the divergent functions of the V-ATPase and the pathophysiology of a4-related symptoms.

Screening of living kidney donors may require scintigraphy to split glomerular filtration rate (GFR). To determine the usefulness of computed tomography (CT) to split GFR, we compared scintigraphy-split GFR to CT-split GFR. We evaluated CT-split GFR as a screening test to detect scintigraphy-split GFR lower than 40 mL/min/1.73 m2/kidney. This was a monocentric retrospective study on 346 potential living donors who had GFR measurement, renal scintigraphy, and CT. We predicted GFR for each kidney by splitting GFR using the following formula: Volume-split GFR for a given kidney = measured GFR*[volume of this kidney/(volume of this kidney + volume of the opposite kidney)]. The same formula was used for length-split GFR. We compared length- and volume-split GFR to scintigraphy-split GFR at donation and with a 4-year follow-up. A better correlation was observed between length-split GFR and scintigraphy-split GFR (r = 0.92) than between volume-split GFR and scintigraphy-split GFR (r = 0.89). A length-split GFR threshold of 45 mL/min/1.73 m2/kidney had a sensitivity of 100 % and a specificity of 75 % to detect scintigraphy-split GFR less than 40 mL/min/1.73 m2/kidney. Both techniques with their respective thresholds detected living donors with similar eGFR evolution during follow-up. Length-split GFR can be used to detect patients requiring scintigraphy. • Excellent correlation between kidney length and scintigraphy predicted GFR • Kidney length screening detects all donors with GFR lower than 40 mL/min/1.73 m 2 • Kidney length screening can replace scintigraphy screening.

Purpose of review: There is consensus that the abnormal retention of sodium by the kidney is the most important pathophysiological event in hypertension. The present review summarizes our current understanding of sodium reabsorption in the distal nephron. Recent findings: The antihypertensive effect of thiazides is thought to be mediated by inhibiting Na+ uptake via the NaCl cotransporter NCC in the distal convoluted tubule. Although it was known that thiazide-sensitive Na⁺ reabsorption in isolated cortical collecting ducts can occur independently of the epithelial Na⁺ channel ENaC, its molecular correlate was unresolved. It was absent in isolated cortical collecting ducts of mice with a targeted disruption of the Na⁺-driven chloride/bicarbonate exchanger NDCBE suggesting that this pathway involves apical Na⁺ uptake into intercalated cells via the Na⁺-driven anion-exchanger NDCBE (SLC4A8). Summary: The finding that SLC4A8-dependent thiazide-sensitive Na⁺ reabsorption occurs in the cortical collecting duct challenges our current model of how thiazides mediate their antihypertensive action and identifies a potentially new target for antihypertensive strategies.

SummaryThe insulin receptor-related receptor (IRR), an orphan receptor tyrosine kinase of the insulin receptor family, can be activated by alkaline media both in vitro and in vivo at pH >7.9. The alkali-sensing property of IRR is conserved in frog, mouse, and human. IRR activation is specific, dose-dependent and quickly reversible and demonstrates positive cooperativity. It also triggers receptor conformational changes and elicits intracellular signaling. The pH sensitivity of IRR is primarily defined by its L1F extracellular domains. IRR is predominantly expressed in organs that come in contact with mildly alkaline media. In particular, IRR is expressed in the cell subsets of the kidney that secrete bicarbonate into urine. Disruption of IRR in mice impairs the renal response to alkali loading attested by development of metabolic alkalosis and decreased urinary bicarbonate excretion in response to this challenge. We therefore postulate that IRR is an alkali sensor that functions in the kidney to manage metabolic bicarbonate excess.

Les mutations inactivatrices du gene de la pendrine sont responsables d’une pathologie associant des troubles de la thyroide et de l’oreille interne. Cependant, il est rapidement apparu apres sa decouverte que l’echangeur d’anions code par ce gene joue un role essentiel dans le rein. Cette breve revue decrit l’evolution de nos connaissances concernant ces differents roles, jusqu’a la tres recente decouverte de l’implication de la pendrine dans la regulation de l’homeostasie du sodium et de la pression arterielle.

Ammonium chloride addition to drinking water is often used to induce metabolic acidosis (MA) in rodents but may also cause mild dehydration. Previous microarray screening of acidotic mouse kidneys showed upregulation of genes involved in renal water handling. Thus, we compared two protocols to induce metabolic acidosis in mice and rats: standard 0.28M NH(4)Cl in drinking water or an equivalent amount of NH(4)Cl in food. Both treatments induced MA in mice and rats. In rats, NH (4)Cl in water caused signs of dehydration, reduced mRNA abundance of the vasopression receptor 2 (V2R), increased protein abundance of the aquaporin water channels AQP2 and AQP3 and stimulated phosphorylation of AQP2 at residues Ser256 and Ser261. In contrast, NH(4)Cl in food induced massive diuresis, decreased mRNA levels of V2R, AQP2, and AQP3, did not affect protein abundance of AQP2 and AQP3, and stimulated phosphorylation at Ser261 but not pSer256 of AQP2. In mice, NH(4)Cl in drinking water stimulated urine concentration, increased AQP2 and V2R mRNA levels, and enhanced AQP2 and AQP3 protein expression with higher levels of AQP2 pSer256 and pSer261. Addition of NH(4)Cl to food, stimulated diuresis, had no effect on mRNA levels of AQP2, AQP3, and V2R, and enhanced only AQP3 protein abundance whereas pSer256-AQP2 and pSer261-AQP2 remained unaltered. Similarly, AQP2 staining was more intense and luminal in kidney from mice with NH(4)Cl in water but not in food. Pendrin, SNAT3 and PEPCK mRNA expression in mouse kidney were not affected by the route of N(4)Cl application. Thus, addition of NH(4)Cl to water or food causes MA but has differential effects on diuresis and expression of mRNAs and proteins related to renal water handling. Moreover, mice and rats respond differently to NH(4)Cl loading, and increased water intake and diuresis may be a compensatory mechanism during MA. It may be necessary to consider these effects in planning and interpreting experiments of NH(4)Cl supplementation to animals.

Objective: To evaluate bone mineral density (BMD), fractures, and vitamin D deficiency in pediatric patients in complete remission of solid tumor; and to identify risk factors for these three abnormalities. Study Design: Data were collected prospectively after completion of cancer treatment. Hormonal and vitamin D deficiencies were treated. The patients were evaluated again 1 year later. Patients: 52 consecutive patients, 30 boys and 22 girls. Among them, 21 completed the second evaluation. Measurements: A clinical examination, nutritional assessment, and laboratory workup were performed. BMD was measured by absorptiometry. Results: Calcium intake was inadequate in 75% of patients and vitamin D reserves were low in 61.5%. BMD was low at the spine in 32.7%, and at the femur in 24% of patients. Spinal and femoral BMD Z-scores correlated significantly with each other. Femoral BMD Z-score showed significant positive correlations with changes in body mass index, urinary calcium/creatinine ratio, and time since treatment completion, and a significant negative correlation with treatment duration. Fractures were noted in 10 patients but were not correlated with BMD. In the 21 re-evaluated patients, no significant improvements were found in calcium intake, vitamin D status, or BMD Z-score. Conclusions: Survivors of childhood solid cancer have high rates of insufficient calcium intake, vitamin D deficiency, low bone mass and fractures.

An inverse relationship exists between urinary tissue kallikrein (TK) excretion and blood pressure in humans and rodents. In the kidney TK is synthesized in large amounts in the connecting tubule and is mainly released into the urinary fluid where its function remains unknown. In the present study mice with no functional gene coding for TK (TK-/-) were used to test whether the enzyme regulates apically expressed sodium transporters. Semiquantitative immunoblotting of the renal cortex revealed an absence of the 70-kDa form of gamma-ENaC in TK-/- mice. Urinary Na+ excretion after amiloride injection was blunted in TK-/- mice, consistent with reduced renal ENaC activity. Amiloride-sensitive transepithelial potential difference in the colon, where TK is also expressed, was decreased in TK-/- mice, whereas amiloride-sensitive alveolar fluid clearance in the lung, where TK is not expressed, was unchanged. In mice lacking the B2 receptor for kinins, the abundance of the 70-kDa form of gamma-ENaC was increased, indicating that its absence in TK-/- mice is not kinin-mediated. Incubation of membrane proteins from renal cortex of TK-/- mice with TK resulted in the appearance of the 70-kDa band of the gamma-ENaC, indicating that TK was able to promote gamma-ENaC cleavage in vitro. Finally, in mouse cortical collecting ducts isolated and microperfused in vitro, the addition of TK in the luminal fluid increased significantly intracellular Na+ concentration, consistent with an activation of the luminal entry of the cation. The results demonstrate that TK, like several other proteases, can activate ENaC in the kidney and the colon.

Recent studies indicate that pendrin, an apical Cl-/HCO3- exchanger, mediates chloride reabsorption in the connecting tubule and the cortical collecting duct and therefore is involved in extracellular fluid volume regulation. The purpose of this study was to test whether pendrin is regulated in vivo primarily by factors that are associated with changes in renal chloride transport, by aldosterone, or by the combination of both determinants. For achievement of this goal, pendrin protein abundance was studied by semiquantitative immunoblotting in different mouse models with altered aldosterone secretion or tubular chloride transport, including NaCl loading, hydrochlorothiazide administration, NaCl co-transporter knockout mice, and mice with Liddle's mutation. The parallel regulation of the aldosterone-regulated epithelial sodium channel (ENaC) was examined as a control for biologic effects of aldosterone. Major changes in pendrin protein expression were found in experimental models that are associated with altered renal chloride transport, whereas no significant changes were detected in pendrin protein abundance in models with altered aldosterone secretion. Moreover, in response to hydrochlorothiazide administration, pendrin was downregulated despite a marked secondary hyperaldosteronism. In contrast, alpha-ENaC was markedly upregulated, and the molecular weight of a large fraction of gamma-ENaC subunits was shifted from 85 to 70 kD, consistent with previous results from rat models with elevated plasma aldosterone levels. These results suggest that factors that are associated with changes in distal chloride delivery govern pendrin expression in the connecting tubule and cortical collecting duct.

Rhesus glycoproteins are a recently discovered family of ammonium transporters and a new branch of the Mep/AMT proteins superfamily that was identified more than 15 years ago in lower organisms and plants. Despite many ex vivo studies showing evidences that Rh glycoproteins can accelerate transmembrane NH3 or NH4+ transfer, their role in normal and disease physiology remains unknown. This review focuses on some of the different studies carried out in animal models to gain insight into Rh glycoprotein function. Immunolocalization studies have added new evidence that this protein family is related to ammonium transport or metabolism in epithelial cells. However, the absence of distal tubular acidosis or hyperammonemia in Rhbg KO mice have raised new questions about the physiological significance of these proteins.

RhBG is a nonerythroid member of the Rhesus (Rh) protein family, mainly expressed in the kidney and belonging to the Amt/Mep/Rh superfamily of ammonium transporters. The epithelial expression of renal RhBG is restricted to the basolateral membrane of the connecting tubule and collecting duct cells. We report here that sorting and anchoring of RhBG to the basolateral plasma membrane require a cis-tyrosine-based signal and an association with ankyrin-G, respectively. First, we show by using a model of polarized epithelial Madin-Darby canine kidney cells that the targeting of transfected RhBG depends on a YED motif localized in the cytoplasmic C terminus of the protein. Second, we reveal by yeast two-hybrid analysis a direct interaction between an FLD determinant in the cytoplasmic C-terminal tail of RhBG and the third and fourth repeat domains of ankyrin-G. The biological relevance of this interaction is supported by two observations. (i) RhBG and ankyrin-G were colocalized in vivo in the basolateral domain of epithelial cells from the distal nephron by immunohistochemistry on kidney sections. (ii) The disruption of the FLD-binding motif impaired the membrane expression of RhBG leading to retention on cytoplasmic structures in transfected Madin-Darby canine kidney cells. Mutation of both targeting signal and ankyrin-G-binding site resulted in the same cell surface but nonpolarized expression pattern as observed for the protein mutated on the targeting signal alone, suggesting the existence of a close relationship between sorting and anchoring of RhBG to the basolateral domain of epithelial cells.

Pendrin (Pds; Slc26A4) is a new anion exchanger that is believed to mediate apical Cl−/HCO3−exchange in type B and non-A-non-B intercalated cells of the connecting tubule and cortical collecting duct. Recently, it has been proposed that this transporter may be involved in NaCl balance and blood pressure regulation in addition to its participation in the regulation of acid-base status. The purpose of our study was to determine the regulation of Pds protein abundance during chronic changes in chloride balance. Rats were subjected to either NaCl, NH4Cl, NaHCO3, KCl, or KHCO3loading for 6 days or to a low-NaCl diet or chronic furosemide administration. Pds protein abundance was estimated by semiquantitative immunoblotting in renal membrane fractions isolated from the cortex of treated and control rats. We observed a consistent inverse relationship between Pds expression and diet-induced changes in chloride excretion independent of the administered cation. Conversely, NaCl depletion induced by furosemide was associated with increased Pds expression. We conclude that Pds expression is specifically regulated in response to changes in chloride balance.

pH dependence of Na + / myo -inositol cotransporters in rat thick limb cells. Background To balance medullary interstitium hypertonicity generated by transepithelial NaCl absorption, medullary thick ascending limb (MTAL) cells accumulate myo -inositol (MI). Expression of Na + -MI cotransporter (SMIT) mRNA in TAL is correlated with the NaCl absorption rate. Our present study aimed to determine the plasma membrane location and functional properties of the Na + -MI cotransporter in MTAL cells. Methods Preparation of basolateral (BLMV) and luminal (LMV) membrane vesicles were simultaneously isolated from purified rat MTAL suspension, and uptake of [ 3 H] myo- inositol ([ 3 H]MI) was used to assess Na + -MI cotransport activity. Results In the presence of an inside-negative membrane potential, imposing an inwardly-directed Na + -gradient versus tetramethylammonium (TMA) stimulated the initial [ 3 H]MI uptake in BLMV and LMV. Phlorizin inhibited Na + gradient-dependent initial [ 3 H]MI uptake in both preparations, with IC 50 values of 565 and 29 μmol/L in BLMV and LMV, respectively. 2-0,C-methylene myo -inositol (MMI), a competitive inhibitor of MI transport, only inhibited the BLMV Na + -MI cotransporter. Phlorizin-sensitive Na + gradient-dependent initial [ 3 H]MI uptake showed Michaelis-Menten kinetics in both preparations, with similar V max but different K m values of 51 and 107 μmol/L in BLMV and LMV, respectively. Finally, BLMV but not LMV Na + -MI cotransporter exhibited a marked pH dependence with sigmoidal patterns of activation, as intravesicular pH (pH i ) was decreased from 8.0 to 6.0 at extravesicular pH (pH e ) 8.0, and as pH e was increased from 6.0 to 8.0 at pH i 6.0. Maximal activation was observed at pH i 6.5 and pH e 7.5. Conclusions In rat MTAL cells, Na + -MI cotransporter activity is present in both BLM and LM, and has markedly different functional properties, indicating the presence of distinct transporters. Basolateral Na + -MI cotransporter activity is maximal at physiological pH values of MTAL cells and interstitium, and a powerful modulation of the transporter activity may be exerted by pH e and pH i .