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Mutations in NKCC2 generate antenatal Bartter syndrome type 1 (type 1 BS), a life-threatening salt-losing nephropathy characterized by arterial hypotension, as well as electrolyte abnormalities. In contrast to the genetic inactivation of NKCC2, inappropriate increased NKCC2 activity has been associated with salt-sensitive hypertension. Given the importance of NKCC2 in salt-sensitive hypertension and the pathophysiology of prenatal BS, studying the molecular regulation of this Na-K-2Cl cotransporter has attracted great interest. Therefore, several studies have addressed various aspects of NKCC2 regulation, such as phosphorylation and post-Golgi trafficking. However, the regulation of this cotransporter at the pre-Golgi level remained unknown for years. Similar to several transmembrane proteins, export from the ER appears to be the rate-limiting step in the cotransporter's maturation and trafficking to the plasma membrane. The most compelling evidence comes from patients with type 5 BS, the most severe form of prenatal BS, in whom NKCC2 is not detectable in the apical membrane of thick ascending limb (TAL) cells due to ER retention and ER-associated degradation (ERAD) mechanisms. In addition, type 1 BS is one of the diseases linked to ERAD pathways. In recent years, several molecular determinants of NKCC2 export from the ER and protein quality control have been identified. The aim of this review is therefore to summarize recent data regarding the protein quality control of NKCC2 and to discuss their potential implications in BS and blood pressure regulation.

Inactivating mutations of kidney Na-K-2Cl cotransporter NKCC2 lead to antenatal Bartter syndrome (BS) type 1, a life-threatening salt-losing tubulopathy. We previously reported that this serious inherited renal disease is linked to the endoplasmic reticulum-associated degradation (ERAD) pathway. The purpose of this work is to characterize further the ERAD machinery of NKCC2. Here, we report the identification of ancient ubiquitous protein 1 (AUP1) as a novel interactor of NKCC2 ER-resident form in renal cells. AUP1 is also an interactor of the ER lectin OS9, a key player in the ERAD of NKCC2. Similar to OS9, AUP1 co-expression decreased the amount of total NKCC2 protein by enhancing the ER retention and associated protein degradation of the cotransporter. Blocking the ERAD pathway with the proteasome inhibitor MG132 or the α-mannosidase inhibitor kifunensine fully abolished the AUP1 effect on NKCC2. Importantly, AUP1 knock-down or inhibition by overexpressing its dominant negative form strikingly decreased NKCC2 polyubiquitination and increased the protein level of the cotransporter. Interestingly, AUP1 co-expression produced a more profound impact on NKCC2 folding mutants. Moreover, AUP1 also interacted with the related kidney cotransporter NCC and downregulated its expression, strongly indicating that AUP1 is a common regulator of sodium-dependent chloride cotransporters. In conclusion, our data reveal the presence of an AUP1-mediated pathway enhancing the polyubiquitination and ERAD of NKCC2. The characterization and selective regulation of specific ERAD constituents of NKCC2 and its pathogenic mutants could open new avenues in the therapeutic strategies for type 1 BS treatment.

Hypoxia stabilizes the transcription factor HIF-1α, which promotes the transcription of many genes essential to adapt to reduced oxygen levels. Besides proline hydroxylation, expression of HIF-1α is also regulated by a range of other posttranslational modifications including phosphorylation by cAMP-dependent protein kinase A (PKA), which stabilizes HIF-1α. We recently demonstrated that MAGED2 is required for cAMP generation under hypoxia and proposed that this regulation may explain the transient nature of antenatal Bartter syndrome (aBS) due to MAGED2 mutations. Consequently, we sought to determine whether hypoxic induction of HIF-1α requires also MAGED2. In HEK293 and HeLa cells, MAGED2 knock-down impaired maximal induction of HIF-1α under physical hypoxia as evidenced by time-course experiments, which showed a signification reduction of HIF-1α upon MAGED2 depletion. Similarly, using cobalt chloride to induce HIF-1α, MAGED2 depletion impaired its appropriate induction. Given the known effect of the cAMP/PKA pathway on the hypoxic induction of HIF-1α, we sought to rescue impaired HIF-1α induction with isoproterenol and forskolin acting upstream and downstream of Gαs, respectively. Importantly, while forskolin induced HIF-1α above control levels in MAGED2-depleted cells, isoproterenol had no effect. To further delineate which PKA subtype is involved, we analyzed the effect of two PKA inhibitors and identified that PKA type II regulates HIF-1α. Interestingly, MAGED2 mRNA and protein were also increased under hypoxia by a cAMP mimetic. Moreover, MAGED2 protein expression also required HIF-1α. Thus, our data provide evidence for reciprocal regulation of MAGED2 and HIF-1α under hypoxia, revealing therefore a new regulatory mechanism that may further explain the transient nature of aBS caused by MAGED2 mutations.

Mutations in the apical Na-K-2Cl co-transporter, NKCC2, cause type I Bartter syndrome (BS1), a life-threatening kidney disease. We have previously demonstrated that the BS1 variant Y998X, which deprives NKCC2 from its highly conserved dileucine-like motifs, compromises co-transporter surface delivery through ER retention mechanisms. However, whether these hydrophobic motifs are sufficient for anterograde trafficking of NKCC2 remains to be determined. Interestingly, sequence analysis of NKCC2 C-terminus revealed the presence of consensus di-acidic (D/E-X-D/E) motifs, 949 EEE 951 and 1019 DAELE 1023 , located upstream and downstream of BS1 mutation Y998X, respectively. Di-acidic codes are involved in ER export of proteins through interaction with COPII budding machinery. Importantly, whereas mutating 949 EEE 951 motif to 949 AEA 951 had no effect on NKCC2 processing, mutating 1019 DAE 1021 to 1019 AAA 1021 heavily impaired complex-glycosylation and cell surface expression of the cotransporter in HEK293 and OKP cells. Most importantly, triple mutation of D, E and E residues of 1019 DAELE 1023 to 1019 AAALA 1023 almost completely abolished NKCC2 complex-glycosylation, suggesting that this mutant failed to exit the ER. Cycloheximide chase analysis demonstrated that the absence of the terminally glycosylated form of 1019 AAALA 1023 was caused by defects in NKCC2 maturation. Accordingly, co-immunolocalization experiments revealed that 1019 AAALA 1023 was trapped in the ER. Finally, overexpression of a dominant negative mutant of Sar1-GTPase abolished NKCC2 maturation and cell surface expression, clearly indicating that NKCC2 export from the ER is COPII-dependent. Hence, our data indicate that in addition to the di-leucine like motifs, NKCC2 uses di-acidic exit codes for export from the ER through the COPII-dependent pathway. We propose that any naturally occurring mutation of NKCC2 interfering with this pathway could form the molecular basis of BS1.

Mutations in MAGED2 cause transient Bartter syndrome characterized by severe renal salt wasting in fetuses and infants, which leads to massive polyhydramnios causing preterm labor, extreme prematurity and perinatal death. Notably, this condition resolves spontaneously in parallel with developmental increase in renal oxygenation. MAGED2 interacts with G-alpha-S (Gαs). Given the role of Gαs in activating adenylyl cyclase at the plasma membrane and consequently generating cAMP to promote renal salt reabsorption via protein kinase A (PKA), we hypothesized that MAGED2 is required for this signaling pathway under hypoxic conditions such as in fetuses. Consistent with that, under both physical and chemical hypoxia, knockdown of MAGED2 in renal (HEK293) and cancer (HeLa) cell culture models caused internalization of Gαs, which was fully reversible upon reoxygenation. In contrast to Gαs, cell surface expression of the β2-adrenergic receptor, which is coupled to Gαs, was not affected by MAGED2 depletion, demonstrating specific regulation of Gαs by MAGED2. Importantly, the internalization of Gαs due to MAGED2 deficiency significantly reduced cAMP generation and PKA activity. Interestingly, the internalization of Gαs was blocked by preventing its endocytosis with dynasore. Given the role of E3 ubiquitin ligases, which can be regulated by MAGE-proteins, in regulating endocytosis, we assessed the potential role of MDM2-dependent ubiquitination in MAGED2 deficiency-induced internalization of Gαs under hypoxia. Remarkably, MDM2 depletion or its chemical inhibition fully abolished Gαs-endocytosis following MAGED2 knockdown. Moreover, endocytosis of Gαs was also blocked by mutation of ubiquitin acceptor sites in Gαs. Thus, we reveal that MAGED2 is essential for the cAMP/PKA pathway under hypoxia to specifically regulate Gαs endocytosis by blocking MDM2-dependent ubiquitination of Gαs. This may explain, at least in part, the transient nature of Bartter syndrome caused by MAGED2 mutations and opens new avenues for therapy in these patients.

Mutations in the apically located kidney Na-K-2Cl cotransporter NKCC2 cause type I Bartter syndrome, a life-threatening kidney disorder. We previously showed that transport from the ER represents the limiting phase in NKCC2 journey to the cell surface. Yet very little is known about the ER quality control components specific to NKCC2 and its disease-causing mutants. Here, we report the identification of Golgi alpha1, 2-mannosidase IA (ManIA) as a novel binding partner of the immature form of NKCC2. ManIA interaction with NKCC2 takes place mainly at the cis-Golgi network. ManIA coexpression decreased total NKCC2 protein abundance whereas ManIA knock-down produced the opposite effect. Importantly, ManIA coexpression had a more profound effect on NKCC2 folding mutants. Cycloheximide chase assay showed that in cells overexpressing ManIA, NKCC2 stability and maturation are heavily hampered. Deleting the cytoplasmic region of ManIA attenuated its interaction with NKCC2 and inhibited its effect on the maturation of the cotransporter. ManIA-induced reductions in NKCC2 expression were offset by the proteasome inhibitor MG132. Likewise, kifunensine treatment greatly reduced ManIA effect, strongly suggesting that mannose trimming is involved in the enhanced ERAD of the cotransporter. Moreover, depriving ManIA of its catalytic domain fully abolished its effect on NKCC2. In summary, our data demonstrate the presence of a ManIA-mediated ERAD pathway in renal cells promoting retention and degradation of misfolded NKCC2 proteins. They suggest a model whereby Golgi ManIA contributes to ERAD of NKCC2, by promoting the retention, recycling, and ERAD of misfolded proteins that initially escape protein quality control surveillance within the ER.

Mutations in Na-K-2Cl co-transporter, NKCC2, lead to type I Bartter syndrome (BS1), a life-threatening kidney disease. Yet, our knowledge of the molecular regulation of NKCC2 mutants remains poor. Here, we aimed to identify the molecular pathogenic mechanisms of one novel and three previously reported missense NKCC2 mutations. Co-immunolocalization studies revealed that all NKCC2 variants are not functional because they are not expressed at the cell surface due to retention in the endoplasmic reticulum (ER). Cycloheximide chase assays together with treatment by protein degradation and mannose trimming inhibitors demonstrated that the defect in NKCC2 maturation arises from ER retention and associated degradation (ERAD). Small interfering RNA (siRNA) knock-down experiments revealed that the ER lectin OS9 is involved in the ERAD of NKCC2 mutants. 4-phenyl butyric acid (4-PBA) treatment mimicked OS9 knock-down effect on NKCC2 mutants by stabilizing their immature forms. Importantly, out of the four studied mutants, only one showed an increased protein maturation upon treatment with glycerol. In summary, our study reveals that BS1 is among diseases linked to the ERAD pathway. Moreover, our data open the possibility that maturation of some ER retained NKCC2 variants is correctable by chemical chaperones offering, therefore, promising avenues in elucidating the molecular pathways governing the ERAD of NKCC2 folding mutants., (© 2021 Wiley Periodicals LLC.)

Mutations in the Na-K-2Cl co-transporter NKCC2 lead to type I Bartter syndrome, a life-threatening kidney disease. We previously showed that export from the ER constitutes the limiting step in NKCC2 maturation and cell surface expression. Yet, the molecular mechanisms involved in this process remain obscure. Here, we report the identification of chaperone stress 70 protein (STCH) and the stress-inducible heat shock protein 70 (Hsp70), as two novel binding partners of the ER-resident form of NKCC2. STCH knock-down increased total NKCC2 expression whereas Hsp70 knock-down or its inhibition by YM-01 had the opposite effect. Accordingly, overexpressing of STCH and Hsp70 exerted opposite actions on total protein abundance of NKCC2 and its folding mutants. Cycloheximide chase assay showed that in cells over-expressing STCH, NKCC2 stability and maturation are heavily impaired. In contrast to STCH, Hsp70 co-expression increased NKCC2 maturation. Interestingly, treatment by protein degradation inhibitors revealed that in addition to the proteasome, the ER associated degradation (ERAD) of NKCC2 mediated by STCH, involves also the ER-to-lysosome-associated degradation pathway. In summary, our data are consistent with STCH and Hsp70 having differential and antagonistic effects with regard to NKCC2 biogenesis. These findings may have an impact on our understanding and potential treatment of diseases related to aberrant NKCC2 trafficking and expression.

Purpose of Review: Antenatal Bartter's syndrome (aBS) is the most severe form of Bartter's syndrome, requiring close follow-up, in particular during the neonatal period, primarily because of prematurity. The recent identification of a novel and very severe form of aBS merits an update on this topic., Recent Finding: Despite the identification of several genes involved in Bartter's syndrome, about 20% of patients clinically diagnosed with aBS remained without genetic explanation for decades. We recently identified mutations in MAGED2 as a cause of an X-linked form of aBS characterized by a very early onset of severe polyhydramnios and extreme prematurity leading to high mortality. Remarkably, all symptoms in surviving patients with MAGE-D2 mutations resolve spontaneously, within weeks after preterm birth. Interestingly, MAGE-D2 affects the expression of the sodium chloride cotransporters NKCC2 and NCC, explaining thereby the severity of the disease. Importantly, a more recent analysis of MAGED2 in a large French cohort of patients with aBS confirmed our data and showed that females can also be affected., Summary: MAGE-D2 is critical for renal salt reabsorption in the fetus, amniotic fluid volume regulation, and maintenance of pregnancy. Most importantly, MAGED2 must be included in the genetic screening of every form of aBS.

Purpose of Review: Antenatal Bartter syndrome (aBS) is a heterogenous disease resulting from defective ion transport in the thick ascending limb of the loop of Henle. Novel insights into the pathophysiology, as well as the recent identification of a novel genetic cause of aBS, merit an update on this topic., Recent Findings: In aBS, severe salt losing is further aggravated by defective salt sensing in the macula densa, where a reduced tubular salt concentration is perceived and glomerular filtration is increased instead of decreased. As patients with aBS come of age, there is an increased incidence of proteinuria and impaired renal function.Moreover, we recently reported a new form of aBS. Indeed, we described a series of nine families in whom pregnancies with male fetuses where complicated by acute polyhydramnios, preterm delivery and with severe but transient polyuria. We identified mutations in melanoma-associated antigen D2 in all study participants and showed, in vivo and in vitro, reduced expression of the furosemide and thiazide sensitive transporters sodium-potassium-2-chloride cotransporter and sodium chloride cotransporter, respectively., Summary: Genetic studies revealed the complexity of ion transport in the thick ascending limb of the loop of Henle and will help to clarify the pathophysiology, which is essential to design new therapies.

Sodium and its associated anions are the major determinant of extracellular fluid volume, and the reabsorption of Na + by the kidney plays a crucial role in long-term blood pressure control. The goal of this study was to investigate the extent to which inhibitors of transepithelial Na + transport (T Na ) along the nephron alter urinary solute excretion and T Na efficiency and how those effects may vary along different nephron segments. To accomplish that goal, we used the multinephron model developed in the companion study (28). That model represents detailed transcellular and paracellular transport processes along the nephrons of a rat kidney. We simulated the inhibition of the Na + /H + exchanger (NHE3), the bumetanide-sensitive Na + -K + -2Cl - transporter (NKCC2), the Na + -Cl - cotransporter (NCC), and the amiloride-sensitive Na + channel (ENaC). Under baseline conditions, NHE3, NKCC2, NCC, and ENaC reabsorb 36, 22, 4, and 7%, respectively, of filtered Na + The model predicted that inhibition of NHE3 substantially reduced proximal tubule T Na and oxygen consumption (Q O 2 ). Whole-kidney T Na efficiency, as reflected by the number of moles of Na + reabsorbed per moles of O 2 consumed (denoted by the ratio T Na /Q O 2 ), decreased by ∼20% with 80% inhibition of NHE3. NKCC2 inhibition simulations predicted a substantial reduction in thick ascending limb T Na and Q O 2 ; however, the effect on whole-kidney T Na /Q O 2 was minor. Tubular K + transport was also substantially impaired, resulting in elevated urinary K + excretion. The most notable effect of NCC inhibition was to increase the excretion of Na + , K + , and Cl - ; its impact on whole-kidney T Na and its efficiency was minor. Inhibition of ENaC was predicted to have opposite effects on the excretion of Na + (increased) and K + (decreased) and to have only a minor impact on whole-kidney T Na and T Na /Q O 2 Overall, model predictions agree well with measured changes in Na + and K + excretion in response to diuretics and Na + transporter mutations., (Copyright © 2016 the American Physiological Society.)

Background: Three pregnancies with male offspring in one family were complicated by severe polyhydramnios and prematurity. One fetus died; the other two had transient massive salt-wasting and polyuria reminiscent of antenatal Bartter's syndrome., Methods: To uncover the molecular cause of this possibly X-linked disease, we performed whole-exome sequencing of DNA from two members of the index family and targeted gene analysis of other members of this family and of six additional families with affected male fetuses. We also evaluated a series of women with idiopathic polyhydramnios who were pregnant with male fetuses. We performed immunohistochemical analysis, knockdown and overexpression experiments, and protein-protein interaction studies., Results: We identified a mutation in MAGED2 in each of the 13 infants in our analysis who had transient antenatal Bartter's syndrome. MAGED2 encodes melanoma-associated antigen D2 (MAGE-D2) and maps to the X chromosome. We also identified two different MAGED2 mutations in two families with idiopathic polyhydramnios. Four patients died perinatally, and 11 survived. The initial presentation was more severe than in known types of antenatal Bartter's syndrome, as reflected by an earlier onset of polyhydramnios and labor. All symptoms disappeared spontaneously during follow-up in the infants who survived. We showed that MAGE-D2 affects the expression and function of the sodium chloride cotransporters NKCC2 and NCC (key components of salt reabsorption in the distal renal tubule), possibly through adenylate cyclase and cyclic AMP signaling and a cytoplasmic heat-shock protein., Conclusions: We found that MAGED2 mutations caused X-linked polyhydramnios with prematurity and a severe but transient form of antenatal Bartter's syndrome. MAGE-D2 is essential for fetal renal salt reabsorption, amniotic fluid homeostasis, and the maintenance of pregnancy. (Funded by the University of Groningen and others.).

Mutations in the renal specific Na-K-2Cl co-transporter (NKCC2) lead to type I Bartter syndrome, a life-threatening kidney disease featuring arterial hypotension along with electrolyte abnormalities. We have previously shown that NKCC2 and its disease-causing mutants are subject to regulation by endoplasmic reticulum-associated degradation (ERAD). The aim of the present study was to identify the protein partners specifically involved in ERAD of NKCC2. To this end, we screened a kidney cDNA library through a yeast two-hybrid assay using NKCC2 C terminus as bait. We identified OS9 (amplified in osteosarcomas) as a novel and specific binding partner of NKCC2. Co-immunoprecipitation assays in renal cells revealed that OS9 association involves mainly the immature form of NKCC2. Accordingly, immunocytochemistry analysis showed that NKCC2 and OS9 co-localize at the endoplasmic reticulum. In cells overexpressing OS9, total cellular NKCC2 protein levels were markedly decreased, an effect blocked by the proteasome inhibitor MG132. Pulse-chase and cycloheximide-chase assays demonstrated that the marked reduction in the co-transporter protein levels was essentially due to increased protein degradation of the immature form of NKCC2. Conversely, knockdown of OS9 by small interfering RNA increased NKCC2 expression by increasing the co-transporter stability. Inactivation of the mannose 6-phosphate receptor homology domain of OS9 had no effect on its action on NKCC2. In contrast, mutations of NKCC2 N-glycosylation sites abolished the effects of OS9, indicating that OS9-induced protein degradation is N-glycan-dependent. In summary, our results demonstrate the presence of an OS9-mediated ERAD pathway in renal cells that degrades immature NKCC2 proteins. The identification and selective modulation of ERAD components specific to NKCC2 and its disease-causing mutants might provide novel therapeutic strategies for the treatment of type I Bartter syndrome., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

This study aims to understand the extent to which modulation of the Na(+)-K(+)-2Cl(-) cotransporter NKCC2 differential splicing affects NaCl delivery to the macula densa. NaCl absorption by the thick ascending limb and macula densa cells is mediated by apical NKCC2. A recent study has indicated that differential splicing of NKCC2 is modulated by dietary salt (Schieβl IM, Rosenauer A, Kattler V, Minuth WW, Oppermann M, Castrop H. Am J Physiol Renal Physiol 305: F1139-F1148, 2013). Given the markedly different ion affinities of its splice variants, modulation of NKCC2 differential splicing is believed to impact NaCl reabsorption. To assess the validity of that hypothesis, we have developed a mathematical model of macula densa cell transport and incorporated that cell model into a previously applied model of the thick ascending limb (Weinstein AM, Krahn TA. Am J Physiol Renal Physiol 298: F525-F542, 2010). The macula densa model predicts a 27.4- and 13.1-mV depolarization of the basolateral membrane [as a surrogate for activation of tubuloglomerular feedback (TGF)] when luminal NaCl concentration is increased from 25 to 145 mM or luminal K(+) concentration is increased from 1.5 to 3.5 mM, respectively, consistent with experimental measurements. Simulations indicate that with luminal solute concentrations consistent with in vivo conditions near the macula densa, NKCC2 operates near its equilibrium state. Results also suggest that modulation of NKCC2 differential splicing by low salt, which induces a shift from NKCC2-A to NKCC2-B primarily in the cortical thick ascending limb and macula densa cells, significantly enhances salt reabsorption in the thick limb and reduces Na(+) and Cl(-) delivery to the macula densa by 3.7 and 12.5%, respectively. Simulation results also predict that the NKCC2 isoform shift hyperpolarizes the macula densa basolateral cell membrane, which, taken in isolation, may inhibit the release of the TGF signal. However, excessive early distal salt delivery and renal salt loss during a low-salt diet may be prevented by an asymmetric TGF response, which may be more sensitive to flow increases.

Hypertension and chronic kidney disease (CKD) are complex traits representing major global health problems. Multiple genome-wide association studies have identified common variants in the promoter of the UMOD gene, which encodes uromodulin, the major protein secreted in normal urine, that cause independent susceptibility to CKD and hypertension. Despite compelling genetic evidence for the association between UMOD risk variants and disease susceptibility in the general population, the underlying biological mechanism is not understood. Here, we demonstrate that UMOD risk variants increased UMOD expression in vitro and in vivo. Uromodulin overexpression in transgenic mice led to salt-sensitive hypertension and to the presence of age-dependent renal lesions similar to those observed in elderly individuals homozygous for UMOD promoter risk variants. The link between uromodulin and hypertension is due to activation of the renal sodium cotransporter NKCC2. We demonstrated the relevance of this mechanism in humans by showing that pharmacological inhibition of NKCC2 was more effective in lowering blood pressure in hypertensive patients who are homozygous for UMOD promoter risk variants than in other hypertensive patients. Our findings link genetic susceptibility to hypertension and CKD to the level of uromodulin expression and uromodulin's effect on salt reabsorption in the kidney. These findings point to uromodulin as a therapeutic target for lowering blood pressure and preserving renal function.

Activation of the Na(+)-K(+)-2Cl(-)-cotransporter (NKCC2) and the Na(+)-Cl(-)-cotransporter (NCC) by vasopressin includes their phosphorylation at defined, conserved N-terminal threonine and serine residues, but the kinase pathways that mediate this action of vasopressin are not well understood. Two homologous Ste20-like kinases, SPS-related proline/alanine-rich kinase (SPAK) and oxidative stress responsive kinase (OSR1), can phosphorylate the cotransporters directly. In this process, a full-length SPAK variant and OSR1 interact with a truncated SPAK variant, which has inhibitory effects. Here, we tested whether SPAK is an essential component of the vasopressin stimulatory pathway. We administered desmopressin, a V2 receptor-specific agonist, to wild-type mice, SPAK-deficient mice, and vasopressin-deficient rats. Desmopressin induced regulatory changes in SPAK variants, but not in OSR1 to the same degree, and activated NKCC2 and NCC. Furthermore, desmopressin modulated both the full-length and truncated SPAK variants to interact with and phosphorylate NKCC2, whereas only full-length SPAK promoted the activation of NCC. In summary, these results suggest that SPAK mediates the effect of vasopressin on sodium reabsorption along the distal nephron.

Mutations in the apical Na-K-2Cl co-transporter, NKCC2, cause type I Bartter syndrome, a life-threatening kidney disease. Yet the mechanisms underlying the regulation of NKCC2 trafficking in renal cells are scarcely known. We previously showed that naturally occurring mutations depriving NKCC2 of its distal COOH-terminal tail and interfering with the (1081)LLV(1083) motif result in defects in the ER exit of the co-transporter. Here we show that this motif is necessary but not sufficient for anterograde trafficking of NKCC2. Indeed, we have identified two additional hydrophobic motifs, (1038)LL(1039) and (1048)LI(1049), that are required for ER exit and surface expression of the co-transporter. Double mutations of (1038)LL(1039) or (1048)LI(1049) to di-alanines disrupted glycosylation and cell surface expression of NKCC2, independently of the expression system. Pulse-chase analysis demonstrated that the absence of the terminally glycosylated form of NKCC2 was not due to reduced synthesis or increased rates of degradation of mutant co-transporters, but was instead caused by defects in maturation. Co-immunolocalization experiments revealed that (1038)AA(1039) and (1048)AA(1049) were trapped mainly in the ER as indicated by extensive co-localization with the ER marker calnexin. Remarkably, among several analyzed motifs present in the NKCC2 COOH terminus, only those required for ER exit and surface expression of NKCC2 are evolutionarily conserved in all members of the SLC12A family, a group of cation-chloride co-transporters that are targets of therapeutic drugs and mutated in several human diseases. Based upon these data, we propose abnormal anterograde trafficking as a common mechanism associated with mutations depriving NKCC2, and also all other members of the SLC12A family, of their COOH terminus.

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

The sodium-independent anion exchanger pendrin is expressed in several tissues including the kidney cortical collecting duct (CCD), where it acts as a chloride/bicarbonate exchanger and has been shown to participate in the regulation of acid-base homeostasis and blood pressure. The renal sympathetic nervous system is known to play a key role in the development of salt-induced hypertension. This study aimed to determine whether pendrin may partly mediate the effects of β adrenergic receptors (β-AR) on renal salt handling. We investigated the regulation of pendrin activity by the cAMP/protein kinase A (PKA) signaling pathway, both in vitro in opossum kidney proximal (OKP) cells stably transfected with pendrin cDNA and ex vivo in isolated microperfused CCDs stimulated by isoproterenol, a β-AR agonist. We found that stimulation of the cAMP/PKA pathway in OKP cells increased the amount of pendrin at the cell surface as well as its transport activity. These effects stemmed from increased exocytosis of pendrin and were associated with its phosphorylation. Furthermore, cAMP effects on the membrane expression and activity of pendrin were abolished by mutating the serine 49 located in the intracellular N-terminal domain of pendrin. Finally, we showed that isoproterenol increases pendrin trafficking to the apical membrane as well as the reabsorption of both Cl(-) and Na(+) in microperfused CCDs. All together, our results strongly suggest that pendrin activation by the cAMP/PKA pathway underlies isoproterenol-induced stimulation of NaCl reabsorption in the kidney collecting duct, a mechanism likely involved in the sodium-retaining effect of β-adrenergic agonists.

Mutations in the electrogenic Cl(-)/H(+) exchanger ClC-5 gene CLCN5 are frequently associated with Dent disease, an X-linked recessive disorder affecting the proximal tubules. Here, we investigate the consequences in Xenopus laevis oocytes and in HEK293 cells of nine previously reported, pathogenic, missense mutations of ClC-5, most of them which are located in regions forming the subunit interface. Two mutants trafficked normally to the cell surface and to early endosomes, and displayed complex glycosylation at the cell surface like wild-type ClC-5, but exhibited reduced currents. Three mutants displayed improper N-glycosylation, and were nonfunctional due to being retained and degraded at the endoplasmic reticulum. Functional characterization of four mutants allowed us to identify a novel mechanism leading to ClC-5 dysfunction in Dent disease. We report that these mutant proteins were delayed in their processing, and that the stability of their complex glycosylated form was reduced, causing lower cell surface expression. The early endosome distribution of these mutants was normal. Half of these mutants displayed reduced currents, whereas the other half showed abolished currents. Our study revealed distinct cellular mechanisms accounting for ClC-5 loss of function in Dent disease., (© 2011 Wiley-Liss, Inc.)

The renal-specific Na-K-2Cl co-transporter, NKCC2, plays a pivotal role in regulating body salt levels and blood pressure. NKCC2 mutations lead to type I Bartter syndrome, a life-threatening kidney disease. Regulation of NKCC2 trafficking behavior serves as a major mechanism in controlling NKCC2 activity across the plasma membrane. However, the identities of the protein partners involved in cell surface targeting of NKCC2 are largely unknown. To gain insight into these processes, we used a yeast two-hybrid system to screen a kidney cDNA library for proteins that interact with the NKCC2 C terminus. One binding partner we identified was SCAMP2 (secretory carrier membrane protein 2). Microscopic confocal imaging and co-immunoprecipitation assays confirmed NKCC2-SCAMP2 interaction in renal cells. SCAMP2 associated also with the structurally related co-transporter NCC, suggesting that the interaction with SCAMP2 is a common feature of sodium-dependent chloride co-transporters. Heterologous expression of SCAMP2 specifically decreased cell surface abundance as well as transport activity of NKCC2 across the plasma membrane. Co-immunolocalization experiments revealed that intracellularly retained NKCC2 co-localizes with SCAMP2 in recycling endosomes. The rate of NKCC2 endocytic retrieval, assessed by the sodium 2-mercaptoethane sulfonate cleavage assay, was not affected by SCAMP2. The surface-biotinylatable fraction of newly inserted NKCC2 in the plasma membrane was reduced by SCAMP2, demonstrating that SCAMP2-induced decrease in surface NKCC2 is due to decreased exocytotic trafficking. Finally, a single amino acid mutation, cysteine 201 to alanine, within the conserved cytoplasmic E peptide of SCAMP2, which is believed to regulate exocytosis, abolished SCAMP2-mediated down-regulation of the co-transporter. Taken together, these data are consistent with a model whereby SCAMP2 regulates NKCC2 transit through recycling endosomes and limits the cell surface targeting of the co-transporter by interfering with its exocytotic trafficking.

Mutations in the anion exchanger pendrin are responsible for Pendred syndrome, an autosomal recessive disease characterized by deafness and goitre. Pendrin is highly expressed in kidney collecting ducts, where it acts as a chloride/bicarbonate exchanger and thereby contributes to the regulation of acid-base homoeostasis and blood pressure. The present study aimed to characterize the intrinsic properties of pendrin. Mouse pendrin was transfected in HEK (human embryonic kidney) 293 and OKP (opossum kidney proximal tubule) cells and its activity was determined by monitoring changes in the intracellular pH induced by variations of transmembrane anion gradients. Combining measurements of pendrin activity with mathematical modelling we found that its affinity for Cl-, HCO3- and OH- varies with intracellular pH, with increased activity at low intracellular pH. Maximal pendrin activity was also stimulated at low extracellular pH, suggesting the presence of both intracellular and extracellular proton regulatory sites. We identified five putative pendrin glycosylation sites, only two of which are used. Mutagenesis-induced disruption of pendrin glycosylation did not alter its cell-surface expression or polarized targeting to the apical membrane and basal activity, but fully abrogated its sensitivity to extracellular pH. The hither to unknown regulation of pendrin by external pH may constitute a key mechanism in controlling ionic exchanges across the collecting duct and inner ear.

Mutations in the apically located Na(+)-K(+)-2Cl(-) co-transporter, NKCC2, lead to type I Bartter syndrome, a life-threatening kidney disorder, yet the mechanisms underlying the regulation of mutated NKCC2 proteins in renal cells have not been investigated. Here, we identified a trihydrophobic motif in the distal COOH terminus of NKCC2 that was required for endoplasmic reticulum (ER) exit and surface expression of the co-transporter. Indeed, microscopic confocal imaging showed that a naturally occurring mutation depriving NKCC2 of its distal COOH-terminal region results in the absence of cell surface expression. Biotinylation assays revealed that lack of cell surface expression was associated with abolition of mature complex-glycosylated NKCC2. Pulse-chase analysis demonstrated that the absence of mature protein was not caused by reduced synthesis or increased rates of degradation of mutant co-transporters. Co-immunolocalization experiments revealed that these mutants co-localized with the ER marker protein-disulfide isomerase, demonstrating that they are retained in the ER. Cell treatment with proteasome or lysosome inhibitors failed to restore the loss of complex-glycosylated NKCC2, further eliminating the possibility that mutant co-transporters were processed by the Golgi apparatus. Serial truncation of the NKCC2 COOH terminus, followed by site-directed mutagenesis, identified hydrophobic residues (1081)LLV(1083) as an ER exit signal necessary for maturation of NKCC2. Mutation of (1081)LLV(1083) to AAA within the context of the full-length protein prevented NKCC2 ER exit independently of the expression system. This trihydrophobic motif is highly conserved in the COOH-terminal tails of all members of the cation-chloride co-transporter family, and thus may function as a common motif mediating their transport from the ER to the cell surface. Taken together, these data are consistent with a model whereby naturally occurring premature terminations that interfere with the LLV motif compromise co-transporter surface delivery through defective trafficking.

Apical bumetanide-sensitive Na(+)-K(+)-2Cl(-) co-transporter, termed NKCC2, is the major salt transport pathway in kidney thick ascending limb. NKCC2 surface expression is subject to regulation by intracellular protein trafficking. However, the protein partners involved in the intracellular trafficking of NKCC2 remain unknown. Moreover, studies aimed at under-standing the post-translational regulation of NKCC2 have been hampered by the difficulty to express NKCC2 protein in mammalian cells. Here we were able to express NKCC2 protein in renal epithelial cells by tagging its N-terminal domain. To gain insights into the regulation of NKCC2 trafficking, we screened for interaction partners of NKCC2 with the yeast two-hybrid system, using the C-terminal tail of NKCC2 as bait. Aldolase B was identified as a dominant and novel interacting protein. Real time PCR on renal microdissected tubules demonstrated the expression of aldolase B in the thick ascending limb. Co-immunoprecipitation and co-immunolocalization experiments confirmed NKCC2-aldolase interaction in renal cells. Biotinylation assays showed that aldolase co-expression reduces NKCC2 surface expression. In the presence of aldolase substrate, fructose 1,6-bisphosphate, aldolase binding was disrupted, and aldolase co-expression had no further effect on the cell surface level of NKCC2. Finally, functional studies demonstrated that aldolase-induced down-regulation of NKCC2 at the plasma membrane was associated with a decrease in its transport activity. In summary, we identified aldolase B as a novel NKCC2 binding partner that plays a key role in the modulation of NKCC2 surface expression, thereby revealing a new regulatory mechanism governing the co-transporter intracellular trafficking. Furthermore, NKCC2 protein expression in mammalian cells and its regulation by protein-protein interactions, described here, may open new and important avenues in studying the cell biology and post-transcriptional regulation of the co-transporter.

Endothelin-1 (ET-1) increases the activity of Na(+)/H(+) exchanger 3 (NHE3), the major proximal tubule apical membrane Na(+)/H(+) antiporter. This effect is seen in opossum kidney (OKP) cells expressing the endothelin-B (ET(B)) and not in cells expressing the endothelin-A (ET(A)) receptor. However, ET-1 causes similar patterns of protein tyrosine phosphorylation, adenylyl cyclase inhibition, and increases in cell [Ca(2+)] in ET(A)- and ET(B)-expressing OKP cells, implying that an additional mechanism is required for NHE3 stimulation by the ET(B) receptor. The present studies used ET(A) and ET(B) receptor chimeras and site-directed mutagenesis to identify the ET receptor domains that mediate ET-1 regulation of NHE3 activity. We found that binding of ET-1 to the ET(A) receptor inhibits NHE3 activity, an effect for which the COOH-terminal tail is necessary and sufficient. ET-1 stimulation of NHE3 activity requires the COOH-terminal tail and the second intracellular loop of the ET(B) receptor. Within the second intracellular loop, a consensus sequence was identified, KXXXVPKXXXV, that is required for ET-1 stimulation of NHE3 activity. This sequence suggests binding of a homodimeric protein that mediates NHE3 stimulation.

Background: Chronic metabolic acidosis leads to an increase in NHE3 activity that is mediated by endothelin-1 (ET-1) expression and activation of the proximal tubule endothelin B receptor. Chronic metabolic acidosis increases preproET-1 mRNA abundance in kidney cortex, but the cell responsible has not been identified., Methods: PreproET-1 mRNA abundance was quantified by competitive reverse transcription-polymerase chain reaction (RT-PCR) on tissue harvested from control rats or rats in which chronic metabolic acidosis was induced by addition of NH(4)Cl to the drinking water., Results: Chronic metabolic acidosis leads to an increase in preproET-1 mRNA expression in kidney cortex, proximal tubules, and glomeruli. The increase in preproET-1 expression correlates with the decrease in blood [HCO3(-)]. ET-1 expression is also increased by acidosis in abdominal aorta, but not in cardiac muscle., Conclusion: In the renal proximal tubule, chronic metabolic acidosis induces an increase in preproET-1 expression, providing a mechanism for autocrine regulation of proximal tubule NHE3 activity. This response is not unique to the proximal tubule cell, but is also not ubiquitous.

Studying the apical Na/H exchanger NHE2 is difficult in the intact thick ascending limb (TAL) because of its weak expression and transport activity compared with the co-expressed NHE3. From a mouse transgenic for a recombinant plasmid adeno-SV(40) (PK4), we developed an immortalized TAL cell line, referred to as MKTAL, which selectively expresses NHE2 protein and activity. The immortalized cells retain the main properties of TAL cells. They have a stable homogeneous epithelial-like phenotype, express SV(40) T antigen and exhibit polarity with an apical domain bearing few microvilli and separated from lateral domains by typical epithelial-type junctional complexes expressing ZO1 protein. Tamm-Horsfall protein is present on the apical membrane. MKTAL cells express NHE2 and NHE1 proteins but not NHE3 and NHE4, whereby NHE2 protein is expressed selectively in the apical domain of the plasma membrane. NHE2 contributed about half of the total Na/H exchange activity. mRNAs for the Na-K-2Cl cotransporter-2 (NKCC2) and the anion exchangers AE2 and AE3 were also present. While acute exposure to NO donors did not alter NHE2 activity, chronic exposure inhibited NHE2 activity selectively and down-regulated NHE2 mRNA abundance. In conclusion, MKTAL cells retain structural and functional properties of their in vivo TAL counterparts and express functional NHE2 protein in the apical membrane, which may be inhibited by NO. Thus, MKTAL cells may be an appropriate model for studying the cellular mechanisms of NHE2 regulation.

The endothelins (ET) are powerful effector agents that control multiple aspects of kidney function. This review will focus on endothelin's effect on proximal tubule H+ secretion. The proximal tubule is responsible for reabsorbing approximately 80% of filtered NaHCO3 by a mechanism mediated by H+ secretion. The major fraction (60-70%) of proximal tubule H+ secretion across the apical membrane is mediated by an amiloride inhibitable Na+/H+ antiporter, while the remaining is mediated by a vaculoar H(+)-ATPase. Molecular, immunocytochemical, and inhibitor sensitivity studies all demonstrate that virtually all proximal tubule apical Na+/H+ activity is mediated by NHE3. Hence, regulation of proximal tubule H+ secretion involves, in most cases, regulation of apical membrane NHE3. We have recently shown that stimulation of NHE3 activity in metabolic acidosis is mediated by endothelin-1 (ET-1) working through the endothelin B (ETB) receptor. ET-1/ETB stimulated antiporter activity is due to an increase in apical membrane NHE3 abundance, achieved by an increase in exocytic insertion of NHE3 into the apical membrane. We have also shown that acid-stimulated NHE3 activity depends on activation of Pyk2, c-Src, MAP kinase, and the immediate early genes c-Fos and c-Jun. This article summarizes these findings and proposes an acid-activated signaling pathway that is responsible for the increase in NHE3 activity in metabolic acidosis.

Background: Regulation of renal transporter expression has been shown to support adaptation of transporter activities in several chronic situations. Basolateral and apical Na/H exchangers (NHE) in medullary thick ascending limb (MTAL) are involved in NH4+ and HCO3+ absorption, respectively. The NH4+ absorption rate in Henle's loop is increased in chronic metabolic acidosis (CMA) and potassium depletion (KD), which may be secondary to the increased NH4+ concentration in luminal fluid and/or to an increased NH4+ absorptive capacity of MTAL. HCO3- absorptive capacity in Henle's loop is increased in CMA and decreased in metabolic alkalosis, but is unchanged in KD despite the presence of metabolic alkalosis. The present study compared the effects of NH4Cl-induced CMA and KD on the expression of basolateral NHE-1 and the effect of KD on the expression of apical NHE-3 in MTAL., Methods: NHE-1 and NHE-3 mRNAs and proteins were assessed by a competitive reverse transcription-polymerase chain reaction (RT-PCR) method and semiquantitative immunoblots, respectively, in MTAL-purified suspensions from rats with CMA and KD., Results: NHE-1 protein abundance was similarly increased (approximately 90%) at two and five weeks of KD, while NHE-1 mRNA amount in MTAL cells was increased at two weeks of KD and returned to normal values by five weeks of KD. In contrast, NHE-1 mRNA and protein abundance did not change in CMA. NHE-3 protein abundance remained unchanged in both two and five weeks of KD, while NHE-3 mRNA was unchanged by two weeks of KD and reduced by approximately 50% at five weeks of KD., Conclusions: The results suggest the following: (1) in KD, where the increased NH4+ concentration of luminal fluid that favors NH4+ absorption is counterbalanced by a decrease in BSC1 expression and activity, the increased NHE-1 expression may support an increased MTAL NH4+ absorptive capacity in CMA, NHE-1 expression is not specifically regulated and remains unchanged, suggesting that the increase in NH4+ concentration in luminal fluid is the main determinant of increased NH4+ absorption in MTAL. (2) In KD, NHE-3 expression did not decrease despite the presence of metabolic alkalosis, in agreement with the unchanged HCO3- absorptive capacity of Henle's loop.

Decreases in blood pH activate NHE3, the proximal tubular apical membrane Na/H antiporter. In cultured renal epithelial cells, activation of the endothelin-B (ET(B)) receptor increases NHE3 activity. To examine the role of the ET(B) receptor in the response to acidosis in vivo, the present studies examined ET(B) receptor-deficient mice, rescued from neonatal lethality by expression of a dopamine beta-hydroxylase promoter/ET(B) receptor transgene (Tg/Tg:ET(B)(-/-) mice). In proximal tubule suspensions from Tg/Tg:ET(B)(+/-) mice, 10(-8) M endothelin-1 (ET-1) increased NHE3 activity, but this treatment had no effect on tubules from Tg/Tg:ET(B)(-/-) mice. Acid ingestion for 7 days caused a greater decrease in blood HCO(3)(-) concentration in Tg/Tg:ET(B)(-/-) mice compared with Tg/Tg:ET(B)(+/+) and Tg/Tg:ET(B)(+/-) mice. Whereas acid ingestion increased apical membrane NHE3 by 42-46% in Tg/Tg:ET(B)(+/+) and Tg/Tg:ET(B)(+/-) mice, it had no effect on NHE3 in Tg/Tg:ET(B)(-/-) mice. In C57BL/6 mice, excess acid ingestion increased renal cortical preproET-1 mRNA expression 2.4-fold and decreased preproET-3 mRNA expression by 37%. On a control diet, Tg/Tg:ET(B)(-/-) mice had low rates of ammonium excretion, which could not be attributed to an inability to acidify the urine, as well as hypercitraturia, with increased titratable acid excretion. Acid ingestion increased ammonium excretion, citrate absorption, and titratable acid excretion to the same levels in Tg/Tg:ET(B)(-/-) and Tg/Tg:ET(B)(+/+) mice. In conclusion, metabolic acidosis increases ET-1 expression, which increases NHE3 activity via the ET(B) receptor.

The present studies examined the effects of chronic NaCl administration and metabolic alkalosis on NHE-3, an apical Na+/H+ exchanger of the rat medullary thick ascending limb of Henle (MTAL). NaCl administration had no effect on NHE-3 mRNA abundance as assessed by competitive RT-PCR, as well as on NHE-3 transport activity estimated from the Na+-dependent cell pH recovery of Na+-depleted acidified MTAL cells, in the presence of 50 microM Hoe-694, which specifically blocks NHE-1 and NHE-2. Two models of metabolic alkalosis were studied, one associated with high sodium intake, i.e., NaHCO3 administration, and one not associated with high sodium intake, i.e., chloride depletion alkalosis (CDA). In both cases, the treatment induced a significant metabolic alkalosis that was associated with a decrease in NHE-3 transport activity (-27% and -25%, respectively). Negative linear relationships were observed between NHE-3 activity and plasma pH or bicarbonate concentration. NHE-3 mRNA abundance and NHE-3 protein abundance, assessed by Western blot analysis, also decreased by 35 and 25%, respectively, during NaHCO3-induced alkalosis, and by 47 and 33%, respectively, during CDA. These studies demonstrate that high sodium intake has per se no effect on MTAL NHE-3. In contrast, chronic metabolic alkalosis, regardless of whether it is associated with high sodium intake or not, leads to an appropriate adaptation of NHE-3 activity, which involves a decrease in NHE-3 protein and mRNA abundance.

Na+/H+ exchangers (NHE) are plasma transmembrane proteins that exchange extracellular Na+ for intracellular H+. Several isoforms of these antiporters belonging to the same gene family have been cloned and four of them (NHE1 to NHE4) are expressed in the kidney. In the kidney, NHEs isoforms display different tubular and membrane (apical vs basolateral) localization and are involved in different functions: regulation of pH and cell volume, NH4+ secretion and NaHCO3 and NaCl reabsorption. NHE3, which is the apical isoform of the proximal tubule and thick ascending limb of Henle, is involved in bicarbonate reabsorption and displays activation during metabolic acidosis. These recent data showing the acid-activation of NHE3 suggest that NHEs isoforms could be involved in the pathogeny of tubular acidosis.