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The thick ascending limb of the loop of Henle (TAL) is the first segment of the distal nephron, extending through the whole outer medulla and cortex, two regions with different composition of the peritubular environment. The TAL plays a critical role in the control of NaCl, water, acid, and divalent cation homeostasis, as illustrated by the consequences of the various monogenic diseases that affect the TAL. It delivers tubular fluid to the distal convoluted tubule and thereby affects the function of the downstream tubular segments. The TAL is commonly considered as a whole. However, many structural and functional differences exist between its medullary and cortical parts. The present review summarizes the available data regarding the similarities and differences between the medullary and cortical parts of the TAL. Both subsegments reabsorb NaCl and have high Na+-K+-ATPase activity and negligible water permeability; however, they express distinct isoforms of the Na+-K+-2Cl−cotransporter at the apical membrane. Ammonia and bicarbonate are mostly reabsorbed in the medullary TAL, whereas Ca2+and Mg2+are mostly reabsorbed in the cortical TAL. The peptidic hormone receptors controlling transport in the TAL are not homogeneously expressed along the cortical and medullary TAL. Besides this axial heterogeneity, structural and functional differences are also apparent between species, which underscores the link between properties and role of the TAL under various environments.

We have recently reported that type A intercalated cells of the collecting duct secrete Na+by a mechanism coupling the basolateral type 1 Na+-K+-2Cl−cotransporter with apical type 2 H+-K+-ATPase (HKA2) functioning under its Na+/K+exchange mode. The first aim of the present study was to evaluate whether this secretory pathway is a target of atrial natriuretic peptide (ANP). Despite hyperaldosteronemia, metabolic acidosis is not associated with Na+retention. The second aim of the present study was to evaluate whether ANP-induced stimulation of Na+secretion by type A intercalated cells might account for mineralocorticoid escape during metabolic acidosis. In Xenopusoocytes expressing HKA2, cGMP, the second messenger of ANP, increased the membrane expression, activity, and Na+-transporting rate of HKA2. Feeding mice with a NH4Cl-enriched diet increased urinary excretion of aldosterone and induced a transient Na+retention that reversed within 3 days. At that time, expression of ANP mRNA in the collecting duct and urinary excretion of cGMP were increased. Reversion of Na+retention was prevented by treatment with an inhibitor of ANP receptors and was absent in HKA2-null mice. In conclusion, paracrine stimulation of HKA2 by ANP is responsible for the escape of the Na+-retaining effect of aldosterone during metabolic acidosis.

The cortical collecting duct (CCD) forms part of the aldosterone-sensitive distal nephron and plays an essential role in maintaining the NaCl balance and acid-base status. The CCD epithelium comprises principal cells as well as different types of intercalated cells. Until recently, transcellular Na+transport was thought to be restricted to principal cells, whereas (acid-secreting) type A and (bicarbonate-secreting) type B intercalated cells were associated with the regulation of acid-base homeostasis. This review describes how this traditional view has been upended by several discoveries in the past decade. A series of studies has shown that type B intercalated cells can mediate electroneutral NaCl reabsorption by a mechanism involving Na+-dependent and Na+-independent Cl−/HCO3−exchange, and that is energetically driven by basolateral vacuolar H+-ATPase pumps. Other research indicates that type A intercalated cells can mediate NaCl secretion, through a bumetanide-sensitive pathway that is energized by apical H+,K+-ATPase type 2 pumps operating as Na+/K+exchangers. We also review recent findings on the contribution of the paracellular route to NaCl transport in the CCD. Last, we describe cross-talk processes, by which one CCD cell type impacts Na+/Cl−transport in another cell type. The mechanisms that have been identified to date demonstrate clearly the interdependence of NaCl and acid-base transport systems in the CCD. They also highlight the remarkable versatility of this nephron segment.

It is now recognized that the metabolic disorders observed in diabetes are not, or not only due to the lack of insulin or insulin resistance, but also to elevated glucagon secretion. Accordingly, selective glucagon receptor antagonists are now proposed as a novel strategy for the treatment of diabetes. However, besides its metabolic actions, glucagon also influences kidney function. The glucagon receptor is expressed in the thick ascending limb, distal tubule, and collecting duct, and glucagon regulates the transepithelial transport of several solutes in these nephron segments. Moreover, it also influences solute transport in the proximal tubule, possibly by an indirect mechanism. This review summarizes the knowledge accumulated over the last 30 years about the influence of glucagon on the renal handling of electrolytes and urea. It also describes a possible novel role of glucagon in the short-term regulation of potassium homeostasis. Several original findings suggest that pancreatic α-cells may express a “potassium sensor” sensitive to changes in plasma K concentration and could respond by adapting glucagon secretion that, in turn, would regulate urinary K excretion. By their combined actions, glucagon and insulin, working in a combinatory mode, could ensure an independent regulation of both plasma glucose and plasma K concentrations. The results and hypotheses reviewed here suggest that the use of glucagon receptor antagonists for the treatment of diabetes should take into account their potential consequences on electrolyte handling by the kidney.

H-K-ATPase type 2 (HKA2), also known as the “nongastric” or “colonic” H-K-ATPase, is broadly expressed, and its presence in the kidney has puzzled experts in the field of renal ion transport systems for many years. One of the most important and robust characteristics of this transporter is that it is strongly stimulated after dietary K+restriction. This result prompted many investigators to propose that it should play a role in allowing the kidney to efficiently retain K+under K+depletion. However, the apparent absence of a clear renal phenotype in HKA2-null mice has led to the idea that this transporter is an epiphenomenon. This review summarizes past and recent findings regarding the functional, structural and physiological characteristics of H-K-ATPase type 2. The findings discussed in this review suggest that, as in the famous story, the ugly duckling of the X-K-ATPase family is actually a swan.

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.

Four of the seven members of the FXYD protein family have been identified as specific regulators of Na,K-ATPase. In this study, we show that FXYD3, also known as Mat-8, is able to associate with and to modify the transport properties of Na,K-ATPase. In addition to this shared function, FXYD3 displays some uncommon characteristics. First, in contrast to other FXYD proteins, which were shown to be type I membrane proteins, FXYD3 may have a second transmembrane-like domain because of the presence of a noncleavable signal peptide. Second, FXYD3 can associate with Na,K- as well as H,K-ATPases when expressed in Xenopusoocytes. However, in situ (stomach), FXYD3 is associated only with Na,K-ATPase because its expression is restricted to mucous cells in which H,K-ATPase is absent. Coexpressed in Xenopusoocytes, FXYD3 modulates the glycosylation processing of the β subunit of X,K-ATPase dependent on the presence of the signal peptide. Finally, FXYD3 decreases both the apparent affinity for Na+and K+of Na,K-ATPase.

Nongastric H,K-ATPases whose catalytic subunits (AL1) encoded by human ATP1AL1 and homologous animal genes comprise the third distinct group within the X,K-ATPase family. No unique nongastric ? has been identified. Precise in situcolocalization and strong association of AL1 with ?1 of Na,K-ATPase was detected in apical membranes of rodent prostate epithelium. In this tissue, ?1NK serves as an authentic subunit of both the Na,K- and nongastric H,K-pumps. Upon expression in Xenopusoocytes the human AL1 can assemble with ?1NK, and more efficiently with gastric ?HK, into functional H,K-pumps. Both AL1? complexes exhibit a similar K-affinity, and their K-transport depends on intra- and extracellular Na. These data provide new evidence that nongastric H,K-ATPase can perform NaK-exchange, and indicate that ? does not significantly affect this ion-pump function. Analysis of human nongastric H,K-ATPase expressed in Sf-21 insect cells revealed that AL1?HK exhibits substantial enzymatic activities in K-free medium and K stimulates, but Na has inhibitory effect on ATP hydrolysis. Thus, although the nongastric H,K-ATPase can function as NaK exchanger, its reaction mechanism is different from that of the Na,K-ATPase. Human nongastric H,K-ATPase is highly sensitive to bufalin, digoxin, and digitoxin, but almost resistant to digoxigenin and ouabagenin.

The FXYD protein family has recently been defined as a result of the search for homologues of the Na,K-ATPase ? subunit, CHIF, and phospholemman in EST and gene data banks. FXYD7 has been seen to have a role as a brain- and isozyme-specific regulator of NaK-ATPase. In this study, the biosynthesis, membrane topology, nature, and role of the processing of FXYD7 are investigated.

The recently defined FXYD protein family contains seven members that are small, single-span membrane proteins characterized by a signature sequence containing an FXYD motif and three other conserved amino acid residues. Until recently, the functional role of FXYD proteins was largely unknown, with the exception of the ? subunit of Na,K-ATPase, which was shown to be a specific regulator of renal ?1-?1 isozymes. We have investigated whether other members of the FXYD family may have a similar role as the ? subunit and have found that CHIF (corticosteroid hormone-induced factor, FXYD4), FXYD7, as well as phospholemman (FXYD1) specifically associate with Na,K-ATPase and preferentially with ?1-? isozymes in native tissues, and produce distinct effects on the transport properties of Na,K-ATPase that are adapted to the physiological demands of the tissues in which they are expressed. These results provide evidence for a unique and novel mode of regulation of Na,K-ATPase by FXYD proteins that involves a tissue-specific expression of an auxiliary subunit of distinct Na,K-ATPase isozymes.

Bufo marinusbladder H-K-ATPase belongs to the Na-K-ATPase and H-K-ATPase subfamily of oligomeric P-type ATPases and is closely related to rat and human nongastric H-K-ATPases. It has been demonstrated that this ATPase transports K+into the cell in exchange for protons and sodium ions, but the stoichiometry of this cation exchange is not yet known. We studied the electrogenic properties of B. marinusbladder H-K-ATPase expressed in Xenopus laevisoocytes. In a HEPES-buffered solution, K+activation of the H-K-ATPase induced a slow-onset inward current that reached an amplitude of ∼20 nA after 1–2 min. When measurements were performed in a solution containing 25 mM HCO3−at a Pco2of 40 Torr, the negative current activated by K+was reduced. In noninjected oocytes, intracellular alkalization activated an inward current similar to that due toB. marinus H-K-ATPase. We conclude that the transport activity of the nongastric B. marinusH-K-ATPase is not intrinsically electrogenic but that the inward current observed in oocytes expressing this ion pump is secondary to intracellular alkalization induced by proton transport.

In oligomeric P2-ATPases such as Na,K- and H,K-ATPases, % subunits play a fundamental role in the structural and functional maturation of the catalytic α subunit. In the present study we performed a tryptophan scanning analysis on the transmembrane α-helix of the Na,K-ATPase %1 subunit to investigate its role in the stabilization of the α subunit, the endoplasmic reticulum exit of α-% complexes, and the acquisition of functional properties of the Na,K-ATPase. Single or multiple tryptophan substitutions in the % subunits transmembrane domain had no significant effect on the structural maturation of α subunits expressed in Xenopusoocytes nor on the level of expression of functional Na,K pumps at the cell surface. Furthermore, tryptophan substitutions in regions of the transmembrane α-helix containing two GXXXG transmembrane helix interaction motifs or a cysteine residue, which can be cross-linked to transmembrane helix M8 of the α subunit, had no effect on the apparent K+affinity of Na,K-ATPase. On the other hand, substitutions by tryptophan, serine, alanine, or cysteine, but not by phenylalanine of two highly conserved tyrosine residues, Tyr40and Tyr44, on another face of the transmembrane helix, perturb the transport kinetics of Na,K pumps in an additive way. These results indicate that at least two faces of the % subunits transmembrane helix contribute to inter- or intrasubunit interactions and that two tyrosine residues aligned in the % subunits transmembrane α-helix are determinants of intrinsic transport characteristics of Na,K-ATPase.

Na,K-ATPase plays a crucial role in cellular ion homeostasis and is the pharmacological receptor for digitalis in man. Nine different human Na,K-ATPase isozymes, composed of 3 α and β isoforms, were expressed in Xenopusoocytes and were analyzed for their transport and pharmacological properties. According to ouabain binding and K+-activated pump current measurements, all human isozymes are functional but differ in their turnover rates depending on the α isoform. On the other hand, variations in external K+activation are determined by a cooperative interaction mechanism between α and β isoforms with α2-β2 complexes having the lowest apparent K+affinity. α Isoforms influence the apparent internal Na+affinity in the order α1 > α2 > α3 and the voltage dependence in the order α2 > α1 > α3. All human Na,K-ATPase isozymes have a similar, high affinity for ouabain. However, α2-β isozymes exhibit more rapid ouabain association as well as dissociation rate constants than α1-β and α3-β isozymes. Finally, isoform-specific differences exist in the K+/ouabain antagonism which may protect α1 but not α2 or α3 from digitalis inhibition at physiological K+levels. In conclusion, our study reveals several new functional characteristics of human Na,K-ATPase isozymes which help to better understand their role in ion homeostasis in different tissues and in digitalis action and toxicity.

The β-subunit of Na,K-ATPase (βNK) interacts with the catalytic α-subunit (αNK) in the ectodomain, the transmembrane, and the cytoplasmic domain. The functional significance of these different interactions was studied by expressing αNK in Xenopusoocytes along with N-terminally modified βNK or with chimeric βNK/βH,K-ATPase (βHK). Complete truncation of the βNK N terminus allows for cell surface-expressed, functional Na,K-pumps that exhibit, however, reduced apparent K+and Na+affinities as assessed by electrophysiological measurements. A mutational analysis suggests that these functional effects are not related to a direct interaction of the β N terminus with the αNK but rather that N-terminal truncation induces a conformational change in another functionally relevant β domain. Comparison of the functional properties of αNK·βNK, αNK·βHK, or αNK·βNK/βHK complexes shows that the effect of the βNK on K+binding is mainly mediated by its ectodomain. Finally, βHK/NK containing the transmembrane domain of βHK produces stable but endoplasmic reticulum-retained αNK·β complexes, while αNK/βHK complexes can leave the ER but exhibit reduced ouabain binding capacity and transport function. Thus, interactions of both the transmembrane and the ectodomain of βNK with αNK are necessary to form correctly folded Na,K-ATPase complexes that can be targeted to the plasma membrane and/or become functionally competent. Furthermore, the β N terminus plays a role in the β-subunit's folding necessary for correct interactions with the α-subunit.